JP7289264B2 - Detection of Colon Tumors by Analysis of Methylated DNA - Google Patents

Description

[Cross reference to related applications]
This application takes precedence over U.S. Provisional Application No. 62/451,327, filed January 27, 2017, and U.S. Provisional Application No. 62/622,107, filed January 25, 2018. each of which is incorporated by reference in its entirety.

Provided herein are techniques related to tumor detection, particularly, but not limited to, methods, compositions, and related uses for detecting tumors, such as colon cancer.

Colorectal cancer remains the second most common cancer in American men and women combined (Siegel R, et al., CA Cancer J Clin 2013;63:11-30). Cancer is amenable to screening because of the underlying biology that progresses from precursor to cancer (Vogelstein B, et al., Science 2013;339:1546-58). Several trials and strategies are supported by evidence and endorsed by guidelines (Levin B, et al., Gastroenterology 2008; 134:1570-95; Rex DK, et al., Am J Gastroenterol 2009; 104: 739-50; Karl J, et al., Clin Gastroenterol Hepatol 2008;6:1122-8). From a social perspective, screening is considered cost-effective (Karl J, et al., Clin Gastroenterol Hepatol 2008; 6:1122-8; Heitman SJ, et al., PLoS Med 2010; 7: e1000370; Parekh M, et al., Aliment Pharmacol Ther 2008;27:697-712; Sharaf RN, et al., Am J Gastroenterol 2013;108:120-32).

Colorectal cancer is caused by the accumulation of genetic and epigenetic changes, and is the basis for analyzing feces for tumor-specific changes (Berger BM, et al., Pathology 2012; 44: 80- 8). Previous large-scale studies of early-generation stool DNA testing in screening settings showed only moderate sensitivity for colorectal cancer and low sensitivity for advanced adenomas (Ahlquist DA, et al. , Ann Intern Med 2008; 149:441-50, W81; Imperiale TF, et al., N Engl J Med 2004; 351:2704-14). Since then, important advances have been introduced, including stabilization buffers (Boynton KA, et al., Clin Chem 2003; 49:1058-65; Zou H, et al., Cancer Epidemiol Biomarkers Prev 2006 15:1115-9), highly discriminative markers (Ahlquist DA, et al., Gastroenterology 2012; 142:248-56; Bardan E, et al., Israel journal of medical sciences 1997; 33:777-80). , a platform with greater analytical sensitivity (Ahlquist DA, et al., Gastroenterology 2012; 142:248-56; Aronchick CA, et al., Gastrointestinal endoscopy 2000; 52:346-52), logistic regression rather than individual marker values Results determination through the use of analytics, and automation.

Although screening reduces colorectal cancer mortality (Mandel JS, et al., N Engl J Med. 1993, 328:1365-71; Hardcastle JD, et al., Lancet. 1996, 348:1472-7; Kronborg O, et al., Scand J Gastroenterol.2004, 39:846-51;Winawer SJ, et al., J Natl Cancer Inst.1993, 85:1311-8;Singh H, et al., JAMA.2006, 295 :2366-73) and the observed reductions were modest (Singh H, et al., JAMA. 2006; 295, 2366-73; Heresbach D, et al., Eur J Gastroenterol Hepatol. 2006, 18:427- 33), more than half of adults in the United States were unscreened (Meissner HI, Cancer Epidemiol Biomarkers Prev. 2006, 15:389-94).

A new method of cancer screening involves assaying for tumor-specific DNA alterations in cancer patient body samples such as feces, serum, and urine (Osborn NK, Ahlquist DA. Gastroenterology 2005; 128:192- 206; Ahlquist DA, et al., Gastroenterology 2000; 119: 1219-27; Ahlquist DA, et al., Gastroenterology 2002; 5;97:1124- 32;Zou H, et al., Cancer Epidemiol Biomarkers Prev 2006;15:1115-9;Zou HZ, Clin Cancer Res 2002;8:188-91;Hoque MO, J Clin Oncol 2005;23:6569-75 Belinsky SA, et al., Cancer Res 2006; 66:3338-44; Itzkowitz SH, et al., Clin Gastroenterol Hepatol 2007; ). Selecting highly accurate markers is important if efficiency and effectiveness are to be achieved in cancer screening. Due to the molecular heterogeneity of colorectal tumors, high detection rates often require panels of markers.

Several methylated genes have been detected in fecal and serum/plasma samples from colon cancer patients (Ahlquist DA, Gastroenterology 2002; 122: Suppl A40; Chen WD, et al., J Natl Cancer Inst 2005; 97: 1124-32;Zou HZ, et al., Clin Cancer Res 2002;8:188-91;Itzkowitz SH, et al., Clin Gastroenterol Hepatol 2007;5:111-7;Petko Z, et al., Clin Cancer Res. 2005;11:1203-9;Muller HM, et al., Lancet 2004;363:1283-5;Leung WK, et al., Clin Chem 2004;50:2179-82;Ebert MP, et al., Gastroenterology 2006 131:1418-30; Grady WM, et al., Cancer Res 2001;61:900-2). Although several methylated genes have been found in most colon cancers, assay results using body fluids remain suboptimal (Ahlquist DA, et al., Gastroenterology 2002; 122: Suppl A40; Chen WD, et al., J Natl Cancer Inst 2005;97:1124-32;Zou H, et al., Cancer Epidemiol Biomarkers Prev 2006;15:1115-9;Zou HZ, Clin Cancer Res 2002;8:1 88- 91; Belinsky SA, et al., Cancer Res 2006;66:3338-44; Itzkowitz SH, et al., Clin Gastroenterol Hepatol 2007;5:111-7; Petko Z, et al., Clin Cancer Res 2005;11:1203-9; Muller HM, et al., Lancet 2004;363:1283-5; Leung WK, et al., Clin Chem 2004; :2179-82; Ebert MP, et al., Gastroenterology 2006;131:1418-30; Grady WM, et al., Cancer Res 2001;61:900-2).

There is a need for more accurate, easy-to-use, and widely distributable tools that improve the efficacy, acceptability, and accessibility of screening.

Herein, techniques relating to tumor detection, in particular, but not exclusively, methods, compositions for detecting pre-malignant and malignant colorectal cancer by analysis of blood and/or plasma samples of a subject, e.g., a patient. Provide technology for products and related uses. Although the technology is described herein, the section headings used are for organizational purposes only and should not be construed as limiting the subject matter in any way.

Provided herein is a panel of tissue-assayed methylated DNA markers that achieves a very high degree of discrimination for colon cancer, while remaining negative in normal colon tissue. This panel can be applied to colorectal cancer screening, for example, using tests with blood or body fluids.

Markers and/or marker panels (e.g., ANKRD13B; CHST2; GRIN2D; JAM3; LRRC4; ZNF304; ZNF568; ZNF671; CNNM1; DOCK2; DTX1; FERMT3; L4; and annotations selected from ZNF671 The chromosomal regions with

As described herein, the technology provides multiple methylated DNA markers and subsets thereof (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) that highly discriminate against colon cancer. , a set of 12 or more markers) are provided. In experiments, selection filters were applied to candidate markers to identify markers that provide high signal-to-noise ratios and low background levels to provide high specificity and selectivity for cancer screening or diagnosis. . For example, as described herein below, the combination of 12 markers and the carcinoembryonic antigen (CEA) protein has a sensitivity of 67.4% (89% cancer) for all cancer plasma samples tested. 60 cases among the examples), and the result was a specificity of 92.6%.

Accordingly, provided herein are techniques relating to methods of screening for colon cancer in a sample obtained from a subject, such methods comprising, for example, assessing the methylation status of a marker in a sample obtained from a subject, methylation marker DNA and identifying the subject as having colon cancer if the methylation status of the marker differs from the methylation status of the marker assayed in the tumor-free subject. GRIN2D; JAM3; LRRC4; OPLAH; SEP9; PDGFD; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; In some embodiments, the technique involves assaying multiple markers, eg, 2-20, preferably 2-14, more preferably 2-12 markers. FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; Including analytics. In preferred embodiments, the assay involves detection of CEA protein.

The technique is not limited to assessing methylation status. In some embodiments, evaluating the methylation status of markers in a sample comprises determining the methylation status of a single base. In some embodiments, assaying the methylation status of a marker in a sample comprises determining the extent of methylation at multiple bases. Further, in some embodiments, the methylation status of the marker includes that the marker is methylation higher than the normal methylation status of the marker, i.e., the methylation status of the marker in the DNA of a tumor-free subject. In some embodiments, the methylation status of the marker comprises lower methylation of the marker than the normal methylation status of the marker. In some embodiments, the methylation status of the marker comprises a different methylation pattern of the marker relative to the normal methylation status of the marker.

In some embodiments, the technology provides a method of creating a record reporting colon tumors in a sample obtained from a subject,
GRIN2D; JAM3; LRRC4; OPLAH; SEP9; PDGFD; PKIA TBX15; TSPYL5; VAV3; FER1L4; and ZNF671.
b) assaying said sample for the amount of a standard marker in said sample;
c) comparing the amount of said at least one methylation marker gene in said sample with the amount of a standard marker, preferably a methylation standard marker, to determine the methylation status of said at least one marker gene in said sample death,
d) creating a record reporting the methylation status of said at least one marker gene in said sample.

Records reporting the methylation status of markers are in no way limited to any particular form of reporting and may include, for example, electronic medical record updates, printed reports, or electronic messages. In some embodiments, the laboratory data obtained during the assay are included in the report, while in some embodiments, a summary of the data or diagnostic results based on the methylation status determined for at least one marker gene are included in the report.

In some embodiments, the sample is assayed for at least two of the markers, preferably at least one methylated marker gene is VAV3; ZNF671; CHST2; selected from the group consisting of GRIN2D, and QKI. FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKI. In preferred embodiments, a subject sample is assayed for the presence of CEA protein.

In some embodiments, the method used to assay comprises obtaining from a subject a sample comprising DNA, and selectively treating the DNA obtained from such sample with unmethylated cytosine residues in the obtained DNA. treatment with a reagent that modifies to generate a modified residue. In preferred embodiments, the reagent comprises a bisulfite reagent.

The method is not limited to a particular size of the methylation marker region to analyze or the number of nucleotides to analyze for methylation status. In some embodiments, assaying the methylation status of a DNA marker in a sample comprises determining the methylation status of one base; Including determining the degree. In some embodiments, the methylation status of the marker includes higher or lower methylation of the marker than the normal methylation status of the marker, although in some embodiments the methylation status of the marker is methylation A different methylation pattern, eg, a different subset of methylated nucleotides in a methylated region of a marker compared to the normal methylation state of the marker.

The technique is not limited to any particular sample type. For example, in some embodiments the sample is a tissue sample, blood sample, serum sample, or sputum sample. In some embodiments, the tissue sample comprises colon tissue.

The technology is not limited to any particular method of assaying DNA samples. For example, in some embodiments, assays include using the polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, separation by mass, and/or target capture. In certain preferred embodiments, the assay involves using the flap endonuclease method. In a particularly preferred embodiment, the sample DNA and/or standard marker DNA are converted with bisulfite and the assay to determine the methylation level of the DNA is methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific PCR, methylation-specific PCR, This is accomplished by techniques including the use of sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, PCR-flap, flap endonuclease, and/or bisulfite genome sequencing PCR.

In some embodiments the oligonucleotides in said mixture comprise reporter molecules, and in preferred embodiments such reporter molecules comprise fluorophores. In some embodiments the oligonucleotide comprises a flap sequence. In some embodiments, the mixture further comprises one or more of a FRET cassette, a FEN-1 endonuclease and a thermostable DNA polymerase, preferably a bacterial DNA polymerase.

In some embodiments, the techniques used involve multiple markers and/or detection of multiple regions of a single marker, using assays that report detection of multiple markers and/or multiple regions of a single marker, using assays that report on a single signal output, e.g., a single fluorochrome. /or detecting multiple regions of a single marker. For example, in some embodiments, assays are configured to report cleavage of flap endonuclease probes specific for multiple different target sites via a single FRET cassette.

Thereafter, in some embodiments, assaying the sample includes amplification reagents to amplify at least two methylated marker DNAs and flap cleavage reagents to perform a flap endonuclease method on the amplified marker DNAs. preparing a reaction mixture comprising:
i) a first primer pair that produces a first amplified region of methylation marker DNA;
ii) a) comprises a sequence complementary to at least a portion of said first amplified region of methylation marker DNA; and b) is not substantially complementary to said first amplified region of methylation marker DNA. a first probe comprising a flap portion having a first flap sequence;
iii) a second primer pair that produces a second amplified region of the methylation marker DNA;
iv) a) comprises a sequence complementary to at least a portion of said second region of methylation marker DNA, and b) is not substantially complementary to said second amplified region of methylation marker DNA. a second probe comprising a flap portion having the first flap sequence;
v) a DNA polymerase;
vi) a flap endonuclease;

In some embodiments, the first amplified region of methylation marker DNA and the second amplified region of methylation marker DNA are amplified from different regions of the same methylation marker gene; , the first amplified region of methylated marker DNA and the second amplified region of methylated marker DNA are amplified from different methylated marker genes. CHST2; CNNM1; GRIN2D; JAM3; LRRC4; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671.

In a preferred embodiment, amplifying at least two methylation marker DNAs comprises amplifying at least three methylation marker DNAs. In such embodiments, the reagents preferably comprise a third primer pair that produces a third amplified region of methylated marker DNA; a third probe comprising a complementary sequence, and b) comprising a flap portion having the same first flap sequence that is not substantially complementary to the third amplified region of the methylated DNA. .

In some embodiments, standard nucleic acids are also assayed. In such embodiments, the reagent further comprises a standard primer pair for generating an amplified region of the standard nucleic acid, a) a sequence complementary to at least a portion of the amplified region of the standard nucleic acid, and b) the standard A standard probe comprising a flap portion having a second flap sequence that is not substantially complementary to the amplified region of the nucleic acid or the first FRET cassette, and a second FRET cassette comprising a sequence complementary to the second flap sequence and may include

Using assays that report detection of multiple markers and/or multiple regions of a single marker to a single signal output, e.g. The techniques for detecting (regions of) are not limited to analysis of methylation, nor to the detection or assay of sample types or markers described above. For example, in some embodiments, the technology provides a method of characterizing any sample (e.g., from a subject) comprising detecting at least one target nucleic acid in the sample, wherein said Said detection of at least one target nucleic acid comprises a reaction mixture comprising an amplification reagent for generating at least two different amplified DNAs and a flap cleaving reagent for performing a flap endonuclease method on the at least two different amplified DNAs. preparing, wherein the reagent is
i) a first primer pair that produces a first amplified DNA;
ii) a) comprises a sequence complementary to a region of said first amplified DNA, and b) comprises a flap portion having a first flap sequence that is not substantially complementary to said first amplified DNA, a first probe;
iii) a second primer pair that produces a second amplified DNA;
iv) a) a flap portion comprising a sequence complementary to a region of said second amplified DNA and b) having said first flap sequence that is not substantially complementary to said second amplified DNA. , a second probe, and
v) a FRET cassette comprising a sequence complementary to said first flap sequence;
vi) a DNA polymerase;
vii) a flap endonuclease;

In some embodiments, the at least two different target DNAs may comprise at least two different marker genes or marker regions in said sample, while in some embodiments, the at least two different target DNAs are contains at least two distinct regions of a single marker gene in Nucleic acids that can be analyzed using the methods disclosed herein are not limited to any particular type of nucleic acid, and include nucleic acids that can be used as targets for amplification in vitro, e.g., by PCR. OK. In some embodiments, one or more of at least one target nucleic acid in the sample is RNA. As mentioned above, the method is not limited to the analysis of two markers or regions, e.g. can be applied to Further, multiple different target nucleic acids of an assay are reported on a first FRET cassette, multiple different targets of the same assay are reported on a second FRET cassette, and multiple different targets of the same assay are reported on a third FRET cassette. Assays may be combined to report cassettes.

The technology also offers kits. For example, in some embodiments, the kit includes a first primer pair that produces a first amplified DNA, a) a sequence complementary to a region of said first amplified DNA, and b) said a) a first probe comprising a flap portion having a first flap sequence that is not substantially complementary to the first amplified DNA; a second primer pair that produces a second amplified DNA; a second probe comprising a sequence complementary to a region of two amplified DNAs, and b) a flap portion having said first flap sequence not substantially complementary to said second amplified DNA; , a FRET cassette comprising a sequence complementary to said first flap sequence, a DNA polymerase and a flap endonuclease.

GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; AH; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; A kit is provided that includes at least one additional oligonucleotide, at least a portion of which specifically hybridizes to. In preferred embodiments, the kit includes an assay that detects CEA protein. In some embodiments the kit comprises at least two additional oligonucleotides, and in some embodiments the kit further comprises a bisulfite reagent.

FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; specifically hybridize. FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; specifically hybridizes to at least one of the 12 oligonucleotides.

In preferred embodiments, the oligonucleotide(s) provided in the kit are selected from one or more of a capture oligonucleotide, a nucleic acid primer pair, a nucleic acid probe, and an invasion oligonucleotide.

In some embodiments, any one of the above kits further comprises a solid support, such as magnetic beads or particles. In preferred embodiments, the solid support comprises one or more capture reagents, eg, oligonucleotides complementary to said one or more marker genes.

The technology also provides compositions. For example, in some embodiments, a technique includes a first primer pair that produces a first amplified DNA, a) a sequence complementary to a region of the first amplified DNA, and b) a first a first probe comprising a flap portion having a first flap sequence that is not substantially complementary to the amplified DNA of; a second primer pair that produces a second amplified DNA; a second probe comprising a sequence complementary to a region of DNA and b) a flap portion having said first flap sequence that is not substantially complementary to a second amplified DNA; A composition is provided comprising a mixture, eg, a reaction mixture, comprising a FRET cassette comprising a sequence complementary to the flap sequence of , a DNA polymerase, and a flap endonuclease. In preferred embodiments, the composition further comprises a first amplified DNA and a second amplified DNA, wherein the first probe is not substantially complementary to the second amplified DNA and the second probe is not substantially complementary to the first amplified DNA. In some embodiments, the composition comprises a primer or probe bound to DNA.

GRIN2D; JAM3; LRRC4; OPLAH; SEP9; H; PDGFD PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; Including a conjugate with a lysing oligonucleotide. Oligonucleotides in the mixture include, but are not limited to, one or more of capture oligonucleotides, nucleic acid primer pairs, hybridization probes, hydrolysis probes, flap method probes, and invasion oligonucleotides. isn't it.

In some embodiments, the target nucleic acids in the mixture are SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, and 136.

In some embodiments, the mixture comprises A bisulfite converted target nucleic acid comprising a nucleic acid sequence selected from the group consisting of 97, 102, 107, 112, 117, 122, 127, 132, and 137.

[definition]
To facilitate understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

Throughout the specification and claims, the following terms have the meanings expressly associated herein, unless the context clearly dictates otherwise. As used herein, the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may. Furthermore, as used herein, the phrase "in another embodiment" does not necessarily refer to a different embodiment, although it may. Accordingly, various embodiments of the invention, as described below, may be readily combined without departing from the scope or spirit of the invention.

Moreover, as used herein, the term “or” is the inclusive operator “or” and is synonymous with the term “and/or” unless the context clearly dictates otherwise. The term “based on” is non-limiting and recognizes that it is based on additional factors not stated unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

In re Herz, 537F. 2d 549,551-52,190 USPQ 461,463 (CCPA 1976), the transitional phrase "consisting essentially of" as used in the claims of this application: Limit the scope of the claims to the materials or steps specified and those that "do not materially affect the basic and novel feature(s)" of the claimed invention. For example, a composition "consisting essentially of" a stated element may be compared to a pure composition, i.e., a composition "consisting of" Even if present, it may be contained at a level such that the hybrid does not alter the function of the composition being described.

As used herein, "methylation" refers to methylation of cytosine at the C5 or N4 positions of cytosine, methylation at the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated DNA because typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA in which the original template was unmethylated or methylated, respectively.

Thus, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base that is not present in a typical recognized nucleotide base. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine does contain a methyl moiety at the 5-position of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide and 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at position 5 of its pyrimidine ring, but for the purposes of this specification, thymine is a typical nucleotide base of DNA, so methylation of thymine when present in DNA not considered a nucleotide.

As used herein, "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

As used herein, "methylation state," "methylation profile," and "methylation status" of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in the nucleic acid molecule. For example, a nucleic acid molecule containing a methylated cytosine is considered methylated (eg, the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that contains no methylated nucleotides is considered unmethylated.

The methylation state of a particular nucleic acid sequence (eg, a genetic marker or DNA region described herein) may indicate the methylation state of every base within that sequence, or a base within that sequence (eg, one (one or more cytosines), or information about the local methylation density within the sequence, with or without precise positional information within the sequence at which the methylation occurs. can show

The methylation status of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of methylated nucleotides at a particular locus of the nucleic acid molecule. For example, the methylation state of a cytosine at nucleotide 7 of a nucleic acid molecule is methylated if the nucleotide present at nucleotide 7 of the nucleic acid molecule is 5-methylcytosine. Similarly, the methylation state of cytosine at nucleotide 7 of the nucleic acid molecule is unmethylated if the nucleotide present at nucleotide 7 of the nucleic acid molecule is cytosine (and not 5-methylcytosine).

Methylation status can optionally be represented or indicated by a "methylation value" (eg, representing methylation frequency, amount, ratio, percentage, etc.). Methylation values can be used, for example, to quantify the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or to compare amplification profiles after a bisulfite reaction, or to compare bisulfite-treated and untreated nucleic acids. It can be generated by sequence comparison of processed nucleic acids. Thus, a value, eg, a methylation value, represents methylation status and can be used as a quantitative indicator of methylation status across multiple copies of a locus. This is of particular use when it is desirable to compare the methylation status of a sample sequence to a threshold or reference value.

As used herein, "methylation frequency" or "methylation rate (%)" refers to the number of instances in which a molecule or locus is methylated relative to the number of instances in which the molecule or locus is unmethylated. .

Methylation status thus represents the methylation status of a nucleic acid (eg, a genomic sequence). Additionally, methylation status refers to the characteristic of a nucleic acid segment at a particular genomic locus that is associated with methylation. Such features include whether there are methylated cytosine (C) residues within this DNA sequence, the location of the methylated C residue(s), the methylated C residue(s) over any particular region of the nucleic acid. and differences in allelic methylation, such as due to differences in allelic origin. The terms "methylation state," "methylation profile," and "methylation status" also refer to the relative concentration, absolute concentration, or pattern of methylated or unmethylated C across any particular region of nucleic acid in a biological sample. Point. For example, if a cytosine (C) residue(s) within a nucleic acid sequence is methylated, this may be referred to as "hypermethylated" or "highly methylated"; If the cytosine (C) residue(s) is unmethylated, it may be called "hypomethylated" or "low methylated." Similarly, cytosine (C) residue(s) within one nucleic acid sequence are methylated when compared to another nucleic acid sequence (e.g., from a different region or from a different individual). If so, the sequence is considered hypermethylated or highly methylated relative to the other nucleic acid sequence. Alternatively, the cytosine (C) residue(s) within the DNA sequence are methylated when compared to another nucleic acid sequence (e.g., from a different region or from a different individual). If not, the sequence is considered hypomethylated or less methylated than the other nucleic acid sequence. Furthermore, as used herein, the term "methylation pattern" refers to a compilation of sites of methylated and unmethylated nucleotides over a region of a nucleic acid. If two nucleic acids have the same or similar number of methylated and unmethylated nucleotides across the region but differ in the positions of the methylated and unmethylated nucleotides, then the methylation frequency or rate is the same. or similar but with different methylation patterns. Sequences are "differentially methylated" if they differ in degree of methylation (e.g., one sequence is more or less methylated than another), frequency, or pattern. or "specific in methylation" or "in different methylation states". The term "specific methylation" refers to a difference in the level or pattern of nucleic acid methylation in a cancer-positive sample as compared to the level or pattern of nucleic acid methylation in a cancer-negative sample. The term can also refer to levels or patterns of differences between patients with and without postoperative cancer recurrence. Specific methylation and individual levels or patterns of DNA methylation, for example, are prognostic and predictive biomarkers once correct cut-offs or predictive features have been defined.

Methylation status frequencies can be used to represent a population of individuals or a sample from a single individual. For example, a nucleotide locus with a methylation state frequency of 50% is methylated in 50% of the examples and unmethylated in 50% of the examples. Such frequencies can be used, for example, to determine the degree to which a nucleotide locus or nucleic acid region of a population of individuals or nucleic acid collection is methylated. Thus, if methylation in a first population or pool of nucleic acid molecules is different than methylation in a second population or pool of nucleic acid molecules, the methylation status frequency of the first population or pool is population or pool of methylation status frequencies. Such frequencies can also be used to reveal, for example, the degree to which a nucleotide locus or nucleic acid region is methylated in a single individual. For example, such frequencies can be used to reveal the degree of methylation or unmethylation of a population of cells from a tissue sample at a nucleotide locus or nucleic acid region.

As used herein, "nucleotide locus" refers to the position of a nucleotide in a nucleic acid molecule. A nucleotide locus of a methylated nucleotide refers to the position of the methylated nucleotide in the nucleic acid molecule.

Typically, methylation of human DNA occurs at dinucleotide sequences that contain adjacent guanines and cytosines, with the cytosine located 5' to the guanine (also called CpG dinucleotide sequences). Although most cytosines within CpG dinucleotides are methylated in the human genome, some cytosines remain unmethylated in specific CpG dinucleotide-rich genomic regions called CpG islands (e.g., Antequera et al. al. (1990) Cell 62:503-514).

As used herein, "CpG islands" refer to G:C-rich regions of genomic DNA that contain a high number of CpG dinucleotides relative to total genomic DNA. A CpG island can be at least 100, 200, or more base pairs in length, where the region has a G:C content of at least 50% and a ratio of observed CpG frequency to predicted CpG frequency of 0.6. However, in some instances, a CpG island can be at least 500 base pairs in length, where the G:C content of the region is at least 55%, and the ratio of observed CpG frequency to predicted CpG frequency is 0.5%. is 65. Measured CpG frequencies relative to predicted CpG frequencies are calculated according to Gardiner-Garden et al (1987) J. Am. Mol. Biol. 196:261-281. For example, the observed CpG frequency relative to the expected CpG frequency can be calculated according to the formula R = (A x B)/(C x D), where R is the ratio of the observed CpG frequency to the expected CpG frequency, and A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation status is typically determined within CpG islands, eg, in promoter regions. However, it will be appreciated that other sequences in the human genome are also susceptible to DNA methylation, such as CpA and CpT (Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237-5242; Salmon and Kaye (1970) Biochim Biophys Acta 204:340-351; Grafstrom (1985) Nucleic Acids Res.13:2827-2842; Nyce (1986) Nucleic Acids Res. ) Biochem.Biophys.Res. Commun. 145:888-894).

As used herein, a "methylation-specific reagent" refers to a reagent that modifies the nucleotides of a nucleic acid molecule relative to the methylation state of such nucleic acid molecule, or a methylation-specific reagent , refers to a compound or composition or other agent that can alter the nucleotide sequence of a nucleic acid molecule to reflect the methylation state of that nucleic acid molecule. Methods of treating nucleic acid molecules with such reagents can include contacting the nucleic acid molecule with the reagent, optionally in combination with additional steps to effect the desired alteration in the nucleotide sequence. Such methods can be applied such that each unmethylated nucleotide (eg, each unmethylated cytosine) is modified to become a different nucleotide. For example, in some embodiments, such reagents deaminate unmethylated cytosine nucleotides to produce deoxyuracil residues. An exemplary reagent is the bisulfite reagent.

The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, bisulfite, or combinations thereof, and as disclosed herein, methylated CpG dinucleotide sequences and unmethylated Refers to those useful in distinguishing from CpG dinucleotide sequences. Said treatment methods are known in the art (eg PCT/EP2004/011715 and WO2013/116375, each of which is incorporated by reference in its entirety). In some embodiments, the bisulfite treatment is performed in the presence of denaturing solvents such as but not limited to n-alkylene glycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or dioxane derivatives. In some embodiments, denaturing solvents are used at concentrations (v/v) of 1% to 35%. In some embodiments, chroman derivatives such as, for example, 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid, or trihydroxybenzoic acid and its derivatives, such as gallic acid (by reference in its entirety The bisulfite reaction is carried out in the presence of a scavenger such as but not limited to (see PCT/EP2004/011715, which incorporates ). In certain preferred embodiments, the bisulfite reaction comprises treatment with ammonium bisulfite, for example as described in WO2013/116375.

Alternatively, altering a nucleic acid nucleotide sequence with a methylation-specific reagent can result in a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide.

The term "methylation assay" refers to any assay that determines the methylation status of one or more CpG dinucleotide sequences within a sequence of nucleic acids.

As used herein, the “sensitivity” of a given marker (or set of markers used together) refers to the number of samples reporting DNA methylation values above the threshold that distinguishes between neoplastic and non-neoplastic samples. Refers to percentage. In some embodiments, a positive is defined as a histologically confirmed tumor reporting a DNA methylation value above a threshold (e.g., a disease-relevant range), and a false negative is below the threshold. Defined as histologically confirmed tumors reporting DNA methylation values (eg, ranges not associated with any disease). Sensitivity values therefore reflect the probability that a given marker's DNA methylation measurement from a known diseased sample falls within a range of disease-related measurements. As defined herein, the clinical significance of a computed sensitivity value represents the probability estimate that a given marker, when applied to a subject with a clinical condition, would detect the presence of that condition. .

As used herein, the “specificity” of a given marker (or set of markers used together) refers to non-neoplastic markers that report DNA methylation values below the threshold that distinguishes neoplastic from non-neoplastic samples. Refers to the percentage of neoplastic samples. In some embodiments, a negative is defined as a histologically confirmed non-neoplastic sample reporting a DNA methylation value below a threshold (e.g., a range not associated with any disease); is defined as a histologically confirmed non-neoplastic sample reporting a DNA methylation value above a threshold (eg, disease-relevant range). Therefore, the specificity value reflects the probability that a given marker's DNA methylation measurement from a known non-neoplastic sample falls within the range of non-disease-related measurements. As defined herein, the clinical significance of the computed specificity value is the probability estimate that a given marker would detect the absence of a clinical condition when applied to a patient who does not have that condition. represents

As used herein, "selected nucleotides" are the four nucleotides that typically occur in nucleic acid molecules (C, G, T, and A for DNA and C, G, U, and A for RNA). which includes methylated derivatives of the typically occurring nucleotide (e.g., if C is the selected nucleotide, both methylated and unmethylated C are included within the meaning of selected nucleotide). ), wherein methylated selected nucleotides specifically refer to nucleotides that are typically methylated, and unmethylated selected nucleotides specifically refer to typically unmethylated forms refers to a nucleotide that occurs in

The term "methylation-specific restriction enzyme" or "methylation-sensitive restriction enzyme" refers to an enzyme that selectively digests nucleic acids depending on the methylation state of its recognition site. For a restriction enzyme that specifically cuts when the recognition site is unmethylated or hemimethylated, cutting does not occur if the recognition site is methylated, or is significantly less efficient. would be low. For a restriction enzyme that specifically cuts if the recognition site is methylated, cutting will not occur or will occur with significantly lower efficiency if the recognition site is not methylated. Methylation-specific restriction enzymes are preferred, whose recognition sequences contain CG dinucleotides (eg, recognition sequences such as CGCG or CCCGGG). In some embodiments, restriction enzymes that do not cut when the cytosine in this dinucleotide is methylated at carbon atom C5 are further preferred.

The term "primer" refers to whether it occurs non-artificially, e.g., as a nucleic acid fragment obtained from a restriction digest, or synthetically produced, which initiates the synthesis of a primer extension product complementary to a nucleic acid template strand. Refers to an oligonucleotide that can act as a starting point for synthesis when placed in conditions (eg, in the presence of an initiator such as a DNA polymerase and a nucleotide, at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands and then used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the initiator. The exact lengths of the primers will depend on many factors, such as temperature, source of primer, and use of the method.

The term "probe" refers to another oligonucleotide of interest, whether occurring non-artificially, such as in a purified restriction digest, or produced synthetically, recombinantly, or by PCR amplification. Refers to an oligonucleotide (eg, a stretch of nucleotides) that is capable of hybridizing. A probe may be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific gene sequences (eg, "capture probes"). Any probe used in the present invention may, in some embodiments, be any detection method including, but not limited to, enzymes (e.g., ELISA and histochemical assays using enzymes), fluorescent systems, radioactive systems, luminescent systems, and the like. It is contemplated that any "reporter molecule" may be labeled detectably in the system. The present invention is not intended to be limited to any particular detection system or label.

The term "target," as used herein, refers to a nucleic acid sought to be sorted out from other nucleic acids, eg, by probe binding, amplification, isolation, capture, and the like. For example, when used with respect to the polymerase chain reaction, "target" refers to the region of nucleic acid to which the primers used in the polymerase chain reaction are bound, and some assays that do not amplify the target DNA, e.g. When used in some embodiments, the target is such that the probe and invasion oligonucleotide (e.g., INVADER oligonucleotide) combine to form an invasion cleavage structure, thereby detecting the presence of the target nucleic acid. including parts. A "segment" is defined as a region of nucleic acid within a target sequence.

The term "marker", as used herein, distinguishes between non-normal cells (e.g. cancer cells) and normal cells, e.g. the presence or absence of marker substances, or status (e.g. methylation status) Refers to a substance (eg, a nucleic acid, or region of a nucleic acid, or a protein) that can be used to perform based on.

As used herein, the term "tumor" refers to a new and abnormal growth of tissue. Tumors may therefore be premalignant or malignant.

The term "tumor-specific marker" as used herein refers to any biological substance or element that can be used to indicate the presence of a tumor. Examples of biological substances include, without limitation, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (eg, cell membranes and mitochondria), and whole cells. In some examples, markers are specific nucleic acid regions (eg, genes, intragenic regions, specific loci, etc.). Regions of nucleic acids that are markers may be referred to, eg, as "marker genes," "marker regions," "marker sequences," "marker loci," and the like.

The term "sample" is used in its broadest sense. In a sense, it refers to animal cells or tissues. In another sense, it refers to sources and specimens or cultures obtained from biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and include liquids, solids, tissues, and gases. Environmental samples include environmental material such as surface material samples, soil samples, water samples, and industrial samples. These examples should not be construed as limiting the sample types applicable to the present invention.

As used herein, the term "patient" or "subject" refers to an organism that is the subject of various tests provided by technology. The term "subject" includes animals, preferably mammals, including humans. In preferred embodiments, the subject is a primate. In an even more preferred embodiment, the subject is human. Further, for diagnostic methods, preferred subjects are vertebrate subjects. Preferred vertebrates are warm-blooded, and preferred warm-blooded vertebrates are mammals. Preferred mammals are most preferably humans. As used herein, the term "subject" includes both human and animal subjects. Accordingly, provided herein are methods for use in animal therapy. As such, the present technology is applicable to mammals such as humans, as well as mammals of critical importance such as the Amur tiger, which are endangered, economically important mammals such as animals raised on farms for human consumption, and and/or provide diagnostics for mammals of social importance to humans, such as animals kept as pets or kept in zoos. Examples of such animals include carnivores such as cats and dogs; swine, including pigs, boars, and wild boars; cattle, bulls, sheep, giraffes, deer, goats, bison; and ruminants and/or ungulates such as camels; pinnipeds; and horses. Accordingly, veterinary diagnostics and treatments are also provided, including but not limited to domestic pigs, ruminants, ungulates, horses (including racehorses), and the like. . The presently disclosed subject matter further includes systems for diagnosing colon cancer in a subject. The system can be provided, for example, as a commercially available kit that can be used to screen for colon cancer risk or make a diagnosis of colon cancer in a subject from whom a biological sample was taken. Exemplary systems provided in accordance with the present technology include assessing the methylation status of the markers described herein.

The term "amplify" or "amplification" in nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of such a polynucleotide, typically from small amounts of a polynucleotide (e.g., a single polynucleotide molecule). initiated, in which case the amplification product or amplicon is generally detectable. Amplification of polynucleotides involves a variety of chemical and enzymatic processes. During the polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, e.g., US Pat. No. 5,494,810, which is incorporated herein by reference in its entirety), the target DNA molecule or template DNA The generation of multiple DNA copies from one or a few copies of a molecule is a form of amplification. Additional types of amplification include allele-specific PCR (see, eg, US Pat. No. 5,639,611, which is incorporated herein by reference in its entirety), assembly PCR (eg, See U.S. Pat. No. 5,965,408, incorporated herein), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594, incorporated herein by reference in its entirety) ), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671, each of which is hereby incorporated by reference in its entirety), intersequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186, each of which is incorporated herein by reference in its entirety), ligation-mediated PCR (e.g., each of which is incorporated herein by reference in its entirety). Guilfoyle, R. et al., Nucleic Acids Research, 25:1854-1858 (1997), which is incorporated herein in its entirety; PCR (see, eg, Herman, et al., (1996) PNAS 93(13)9821-9826, which is incorporated herein by reference in its entirety), miniprimer PCR, multiplex ligation-dependent probe amplification methods (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12):e57, which is hereby incorporated by reference in its entirety), multiplex PCR (e.g., each of which Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156; Ballabio, et al., (1990) Human Genetics 84(6) 571-573; al., (2008) BMC Genetics 9:80), nested PCR, overlap extension PCR (e.g., Higuchi, et al., (1988) Nucleic Acids Research 16, which is hereby incorporated by reference in its entirety). (15) 7351-7367), real-time PCR (eg, Higuchi, et al., each of which is hereby incorporated by reference in its entirety). , (1992) Biotechnology 10:413-417; Higuchi, et al. , (1993) Biotechnology 11:1026-1030), reverse transcription PCR (eg, Bustin, SA (2000) J. Molecular Endocrinology 25:169-, which is hereby incorporated by reference in its entirety). 193), solid-phase PCR, thermal asymmetric interlaced PCR, and touchdown PCR (e.g., Don, et al., Nucleic Acids Research (1991) 19, each of which is hereby incorporated by reference in its entirety). (14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485. It is not limited. Amplification of polynucleotides can also be accomplished using digital PCR (see, for example, Kalinina, et al., Nucleic Acids Research. 25; 1999-2004, each of which is hereby incorporated by reference in its entirety). Vogelstein and Kinzler, Proc Natl Acad Sci USA.96;9236-41, (1999); International Patent Publication No. WO05023091A2; U.S. Patent Application Publication No. 20070202525).

The term "polymerase chain reaction" ("PCR") describes a method for enriching a segment of a target sequence in a mixture of genomic or other DNA or RNA without cloning or purification. B. Mullis, US Pat. Nos. 4,683,195, 4,683,202, and 4,965,188. This method of amplifying a target sequence involves introducing a large excess of two oligonucleotide primers containing the desired target sequence into a DNA mixture, followed by a series of precise thermal cyclings in the presence of a DNA polymerase. It consists of Each of the two primers is complementary to a respective strand of the double-stranded target sequence. Amplification is carried out by denaturing the mixture and then annealing the primers to their complementary sequences within the target molecule. After annealing, the primers are extended using a polymerase so that a new pair of complementary strands is formed. The steps of denaturation, primer annealing, and polymerase extension are repeated many times (i.e., denaturation, annealing and extension constitute one "cycle", and the number of "cycles" can be many) to obtain the desired target. Segments can be obtained in which the sequences are enriched and amplified. The length of the amplified segment of the desired target sequence is determined by the position of the primers relative to each other, so the length is a manageable parameter. Because the steps are iterative, this method is called the "polymerase chain reaction" ("PCR"). Because the desired amplified segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are referred to as "PCR amplification products" and also as "PCR products" or "amplicons." Those skilled in the art will appreciate that the term "PCR" encompasses many variations of the originally described method using, for example, real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single-primer and arbitrary-prime PCR. Let's understand.

As used herein, the term "nucleic acid detection method" refers to any method that determines the nucleotide composition of a nucleic acid of interest. Nucleic acid detection methods include DNA sequencing methods, probe hybridization methods, structure-specific cleavage methods (e.g., the INVADER method (Hlogic, Inc.) (e.g., U.S. Pat. Nos. 5,846,717, 5,985). , 557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292. (1999), Hall et al., PNAS, USA, 97:8272 (2000), and US2009/0253142, each of which is hereby incorporated by reference in its entirety for all purposes) enzymatic cleavage of mismatches (e.g., Variagenics U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, which are incorporated herein by reference in their entirety); ); polymerase chain reaction (PCR), as described above; branched hybridization methods (e.g., Chiron U.S. Pat. Nos. 5,849,481, 5,710,264, which are incorporated herein by reference in their entireties) , Nos. 5,124,246, and 5,624,802); rolling circle reproductions (e.g., U.S. Pat. 183,960, and 6,235,502); NASBA (e.g., U.S. Pat. No. 5,409,818, which is incorporated herein by reference in its entirety); US Pat. No. 6,150,097, incorporated herein in its entirety); E-Sensor Method (Motorola US Pat. 6,221,583, 6,013,170, and 6,063,573); cycling probe methods (e.g., U.S. Patent Nos. 5,403, which are incorporated herein by reference in their entirety); 711, 5,011,769, and 5,660,988); the Dade Behring signal amplification method (e.g., U.S. Pat. No. 6,121,001, incorporated herein by reference in its entirety); , 6,110,677, 5,914,230, 5,882,867, and 5,792,614); ligase chain reactions (eg, Baranay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization methods (eg, US Pat. No. 5,288,609, which is incorporated herein by reference in its entirety). not something.

In some embodiments, the target nucleic acid is amplified (eg, by PCR) and an invasion cleavage method is simultaneously used to detect the amplified nucleic acid. Assays adapted for performing detection methods (eg, invasion cleavage methods) in combination with amplification methods are described in U.S. Pat. No. 9,096,893, herein incorporated by reference in its entirety for all purposes. incorporated into the book. Additional detection configurations that combine amplification and invasion cleavage methods, called the QuARTS method, are described, for example, in U.S. Pat. Nos. 8,361,720, Nos. 8,715,937; 8,916,344; and 9,212,392. As used herein, the term "invasion cleavage structure" includes i) a target nucleic acid, ii) an upstream nucleic acid (e.g., an invasion oligonucleotide or "INVADER" oligonucleotide), and iii) a downstream nucleic acid (e.g., a probe). Refers to a cleavage structure in which the upstream and downstream nucleic acids anneal to a contiguous region of the target nucleic acid, and between the duplex formed between the downstream and target nucleic acids and the 3' portion of the upstream nucleic acid. Duplicates are formed. Overlap occurs where one or more bases from the upstream and downstream nucleic acids occupy the same position relative to a base of the target nucleic acid, and the overlapping base(s) of the upstream nucleic acid are complementary to the target nucleic acid. or not, and whether those bases are natural bases or non-natural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps with the downstream duplex is disclosed, for example, in US Pat. No. 6,090,543, which is hereby incorporated by reference in its entirety. A chemical moiety other than a base, such as an aromatic ring structure in which In some embodiments, one or more of the nucleic acids may be attached to each other, e.g., via covalent bonds, such as nucleic acid stem-loops, or via non-nucleic acid chemical bonds (e.g., multiple carbon chains). . As used herein, the term "flap endonuclease method" includes the "INVADER" invasion cleavage method and the QuARTS method as described above.

The term "probe oligonucleotide" or "flap oligonucleotide" when used with respect to the flap method refers to an oligonucleotide that interacts with a target nucleic acid to form a cleavage structure in the presence of an invasion oligonucleotide.

The term "invasion oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid at positions adjacent to the hybridization region of the probe and target nucleic acid, wherein the 3' end of the invasion oligonucleotide It includes a portion (eg, a chemical moiety, or one or more nucleotides) that overlaps with the hybridization region. The nucleotides at the 3' end of the invasion oligonucleotide may or may not base pair with nucleotides in the target. In some embodiments, the invasion oligonucleotide contains at its 3' end a sequence that is substantially identical to a sequence located at the 5' end of the portion of the probe oligonucleotide that anneals to the target strand.

The term "flap endonuclease" or "FEN" as used herein refers to a class of nucleolytic enzymes, typically 5' nucleases, which displace another strand of nucleic acid. DNA having a duplex containing a 5' overhang, or flap, on the other single strand (e.g., resulting in overlapping nucleotides at the junction between the single and double stranded DNA) Acts as a structure-specific endonuclease on structures. FEN catalyzes hydrocleavage of phosphodiester bonds at the junctions of single- and double-stranded DNA, liberating overhanging ends, or flaps. Flap endonucleases have been described by Ceska and Savers (Trends Biochem. Sci. 1998 23:331-336) and Liu et al. outlined. FENs may be individual enzymes, multi-subunit enzymes, or may exist as the activity of separate enzymes or protein complexes (eg, DNA polymerases).

A flap endonuclease may be thermostable. For example, the FEN-1 flap endonucleases of well-documented thermophilic organisms are typically thermostable. As used herein, the term "FEN-1" refers to a non-polymerase flap endonuclease from eukaryotic or archaeal organisms. See, for example, WO02/070755, and Kaiser M. et al., which are incorporated herein by reference in their entirety for all purposes. W. , et al. (1999)J. Biol. Chem. , 274:21387.

As used herein, the term "cleavage flap" refers to the single-stranded oligonucleotide that is the cleavage product of the flap method.

The term "cassette" when used in reference to a flap cleavage reaction, e.g., in the primary or first cleavage structure formed by the flap cleavage method, generates a detectable signal in response to cleavage of the flap or probe oligonucleotide. It refers to an oligonucleotide or combination of oligonucleotides that are arranged in a similar manner. In a preferred embodiment, the cassette hybridizes with a non-target cleavage product generated by cleavage of the flap oligonucleotide to form a second overlapping cleavage structure, after which such cassette is conjugated to the same enzyme, eg, FEN- 1 endonuclease.

In some embodiments, the cassette comprises a single strand comprising a hairpin portion (i.e., a region where under reaction conditions a portion of the cassette oligonucleotide hybridizes to a second portion of the same oligonucleotide to form a duplex). is an oligonucleotide. In other embodiments, the cassette comprises at least two oligonucleotides containing complementary portions capable of forming a duplex under reaction conditions. In preferred embodiments, the cassette includes a label, eg, a fluorophore. In a particularly preferred embodiment, the cassette contains a labeling moiety that produces a FRET effect. In such embodiments, the cassette may be referred to as a "FRET cassette." For example, US patent application Ser. See also International Application No. PCT/US16/58875 filed October 26, 2016.

As used herein, when the phrase "not substantially complementary" is used with respect to a flap or arm of a probe, the flap portion is capable of binding a nucleic acid sequence, e.g., a target, under specified annealing or stringent conditions. Sufficiently non-complementary to not selectively hybridize with nucleic acid or amplified DNA, and includes the terms "substantially non-complementary" and "completely non-complementary."

The term "complementary," as used herein, means that the primers or probes have sufficient complementarity to selectively hybridize to a target nucleic acid sequence under, for example, specified annealing or stringent conditions. Complementary means and encompasses the terms "substantially complementary" and "fully complementary."

As used herein, the term "FRET" means that moieties (e.g., fluorophores) transfer energy, e.g., between moieties or from a fluorophore to a non-fluorophore (e.g., a quencher molecule). Refers to fluorescence resonance energy transfer, a process. In some situations, FRET involves an excited donor fluorophore transferring energy to a lower energy acceptor fluorophore via a short-range (eg, about 10 nm or less) dipole-dipole interaction. used. In other situations, FRET uses the loss of fluorescence energy from the donor and the increase in fluorescence at the acceptor fluorophore. In yet another form of FRET, energy can be exchanged from an excited donor fluorophore to a non-fluorescent molecule (eg, a "dark" quencher molecule). FRET is known to those of skill in the art and has been described (e.g., Stryer et al., 1978, Ann. Rev. Biochem., 47:819, each of which is hereby incorporated by reference in its entirety; Selvin, 1995, Methods Enzymol., 246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res 573, 103-110).

In an exemplary flap detection method, an invasion oligonucleotide and a flap oligonucleotide are hybridized to a target nucleic acid to create a first complex having an overlap as described above. An unpaired "flap" is included on the 5' end of the flap oligonucleotide. The first complex is the substrate for a flap endonuclease, eg, FEN-1 endonuclease, which cleaves the flap oligonucleotide and releases the 5' flap portion. In the secondary reaction, the liberated 5' flap product is used as an invasion oligonucleotide on the FRET cassette, again creating a structure that is recognized by the flap endonuclease and causing the FRET cassette to be cleaved. When the fluorophore and quencher are separated by cleavage of the FRET cassette, a detectable fluorescent signal above background fluorescence is generated.

As used herein, the term "real-time" when used in reference to detection of nucleic acid amplification or signal amplification is the detection of product or signal accumulation in a reaction while the reaction is in progress, e.g. Point to measurement. Such detection or measurement may be performed continuously, or while the amplification reaction is in progress, or a combination thereof. For example, in the polymerase chain reaction, detection (e.g., detection of fluorescence) may occur during all or part of thermal cycling, or may occur transiently at one or more points during one or more cycles. may be performed. In some embodiments, real-time detection of PCR or QuARTS reactions is achieved by determining fluorescence levels at the same point (e.g., cycle time point, or cycle temperature step) in each cycle, or in each of multiple cycles. be done. Real-time detection of amplification is also referred to as "during" amplification reaction detection.

As used herein, the term "quantitative amplification data set" refers to data obtained during quantitative amplification of a target sample, eg, target DNA. For quantitative PCR or QuARTS methods, a quantitative amplification data set is a collection of fluorescence values obtained during amplification, eg, multiple thermal cycles or all thermal cycles. Data for quantitative amplification are not limited to collecting data at specific points in the reaction, fluorescence may be measured at discrete points in each cycle or continuously throughout each cycle.

As used herein, the abbreviations "Ct" and "Cp", when used with respect to data collected during real-time PCR and PCR+INVADER methods, indicate that the signal (e.g., fluorescent signal) is a positive signal. refers to the cycle crossing the threshold of Various methods have been used to calculate thresholds for use as determinants of signal versus concentration, and the value is commonly referred to as the "crossing threshold" (Ct) or "crossing point" (Cp). is represented by In embodiments of the methods presented herein, either Cp or Ct values are used for real-time signal analysis for determining the percentage of mutant and/or non-mutant components in an assay or sample. You may

As used herein, the term "control," when used in connection with the detection or analysis of nucleic acids, refers to a known characteristic for use in comparison to an experimental target (e.g., nucleic acid of unknown concentration). It refers to a nucleic acid having (eg, known sequence, known copy number per cell). Controls can be endogenous, preferably invariant genes to which the test or target nucleic acid of the assay can be normalized. Such normalization adjusts for sample-to-sample variability that may occur, for example, in sample processing, assay efficiency, etc., and allows accurate comparison of sample-to-sample data. Genes utilized for normalization of nucleic acid detection methods in human samples include, for example, β-actin, ZDHHC1, and B3GALT6 (see, for example, US Patent Application No. 14, each incorporated herein by reference). /966,617 and 62/364,082).

A control may be an external control. For example, in quantitative assays such as qPCR, QuARTS, etc., a "calibrator" or "calibration control" is a nucleic acid of known sequence, e.g., identical in sequence to a portion of an experimental target nucleic acid, Nucleic acid of known concentration or concentration series (eg serially diluted control target for calibration curve generation in quantitative PCR). Typically, calibration controls are analyzed using the same reagents and reaction conditions as used for experimental DNA. In certain embodiments, calibrator measurements are performed concurrently with experimental assays, eg, on the same thermal cycler. In a preferred embodiment, multiple calibrators may be included in a single plasmid to facilitate obtaining equimolar amounts of different calibrator sequences. In particularly preferred embodiments, the plasmid calibrator is digested, eg, with one or more restriction enzymes, to release the calibrator portion from the plasmid vector. See, for example, WO2015/066695, which is incorporated herein by reference. In some embodiments, the calibrator DNA is synthetic, as described, for example, in US patent application Ser. No. 15/105,178, incorporated herein by reference.

As used herein, "ZDHHC1" is located in human DNA (16q22.1) on Chr16 and encodes a protein characterized as a zinc finger belonging to the DHHC palmitoyltransferase family, DHHC-type containing 1 refers to the gene that

As used herein, the term "process control" means that a sample prior to extraction of measurable target DNA after extraction can be evaluated for process effectiveness and to determine the success or failure of the method. Refers to an exogenous molecule, eg, an exogenous nucleic acid, that is added to. The nature of the process control nucleic acid used will generally depend on the type of assay and substance to be measured. For example, if the assay used is for the detection and/or quantification of double-stranded DNA or mutations contained therein, a double-stranded DNA process control is typically added to the pre-extracted sample. Similarly, for assays that monitor mRNA or microRNA, the process controls used are typically RNA transcripts or synthetic RNA. For example, U.S. Patent Application No. 62/364,049, filed July 19, 2016, which is incorporated herein by reference and describes using zebrafish DNA as a process control for human samples. See No.

As used herein, the term "zebrafish DNA" refers to DNA isolated from or to DNA of Danio rerio, as opposed to bulk "fish DNA" (e.g., purified salmon DNA). Refers to DNA that has been produced in vitro (eg, enzymatically, synthetically) to have the nucleotide sequence found. In a preferred embodiment, the zebrafish DNA is methylated DNA and is added as a detectable control DNA, eg, process control, to verify DNA recovery through each step of sample processing. In particular, zebrafish DNA comprising at least a portion of the RASSF1 gene is utilized as a process control, eg, for human samples, as described in US Patent Application No. 62/364,049.

As used herein, the term “fish DNA” is different from zebrafish DNA, for example, large amounts of DNA isolated from fish, as described in US Pat. No. 9,212,392. refers to the (eg, genomic) DNA of the Bulk purified fish DNA is commercially available, for example in the form of cod and/or herring sperm DNA (Roche Applied Science, Mannheim, Germany) or salmon DNA (USB/Affymetrix).

As used herein, the terms "particles" and "beads" are used interchangeably, and the terms "magnetic particles" and "magnetic beads" are used interchangeably to refer to particles or beads that respond to a magnetic field. . Typically magnetic particles are materials that do not have a magnetic field but form a magnetic dipole when exposed to a magnetic field, e.g. contains materials that are not magnetic per se. The term "magnetic" as used in this context includes paramagnetic or superparamagnetic materials. As used herein, the term "magnetic" also encompasses temporary magnetic materials, such as ferromagnetic or ferrimagnetic materials with low Curie temperatures, such temporary magnetic materials Silica magnetic particles containing such materials are paramagnetic in the temperature range used according to the present method for the isolation of biological substances.

As used herein, the term "kit" refers to any delivery system for delivering substances. In reaction assays, such delivery systems include reaction reagents (e.g., oligonucleotides, enzymes, etc. in suitable containers) and/or supporting materials (e.g., buffers, assays, etc.) from one location to another. Includes systems that allow storage, transport, or delivery of instructions for performance, etc.). For example, kits include one or more enclosures (eg, boxes) containing relevant reaction reagents and/or supporting materials. As used herein, the term "subdivided kit" refers to a delivery system comprising two or more separate containers containing subdivided all components of the kit. The containers may be delivered together or separately to the intended recipient. For example, a first container may contain an enzyme for use in an assay and a second container may contain an oligonucleotide.

As used herein, the term "system" refers to a collection of articles for a particular purpose. In some embodiments, the article includes instructions for use, eg, information provided on the article, on paper, or in a recordable medium (eg, DVD, CD, flash drive, etc.). In some embodiments, the instructions direct the user to go to an online location, eg, a website.

As used herein, the term "information" refers to any collection of facts or data. When used in reference to information stored or processed using a computer system(s), including but not limited to the Internet, the term "information" refers to any form (e.g., analog, digital, optical). ) refers to any data stored in As used herein, the term "information about a subject" refers to facts or data relating to a subject (eg, human, plant, or animal). The term "genomic information" refers to information related to the genome, including but not limited to nucleic acid sequences, genes, methylation rates, allele frequencies, RNA expression levels, protein expression, phenotypes correlated with genotypes, etc. . "Allele frequency information" refers to facts or data relating to allele frequency, including allele identity, statistical correlations between the presence of alleles and characteristics of subjects (e.g., human subjects), Examples include, but are not limited to, the presence or absence of the allele, the likelihood (%) of the presence of the allele in individuals with one or more specific characteristics, and the like.

Figures 1A-1AG show schematic representations of unconverted and bisulfite-converted forms of marker target regions. Flap method primers and probes for detection of bisulfite-converted target DNA are shown. Figures 2A-2H provide a table of nucleic acid sequences and corresponding SEQ ID NOs. 3A-3L provide tables showing data and results from the assay of Example 2. FIG. 4A-4E provide tables showing data and results from the assay of Example 2. FIG. FIG. 5 provides a schematic showing a combined PCR and invasion cleavage assay (“PCR-flap method”), such as the QuARTS method, in which three different regions of a target nucleic acid, such as a methylation marker, are Amplification with primer pairs specific for each of the different regions, but in the presence of different flap probes, each flap probe specific for one of the different regions, but each flap arm The sequences are identical. Flap sends all reports to the same FRET cassette and generates fluorescent signal from the same fluorophore during each PCR-Flap procedure.

Provided herein are techniques relating to the selection and use of nucleic acid markers for use in assays to detect and quantify DNA, eg, methylated DNA. In particular, the technology relates to using methylation assays for colon cancer detection.

In the detailed description of various embodiments herein, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will understand that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Moreover, those skilled in the art will appreciate that the specific order in which the method is presented and performed is exemplary and that such order may be varied while remaining within the spirit and scope of the various embodiments disclosed herein. It can be readily understood that it is intended to be

In some embodiments, analyzing the target DNA comprises analyzing multiple different DNAs in a single reaction. Typical instrumentation for real-time detection of amplification reactions can only detect and quantify 3-5 fluorochromes at a time. This is mainly because when many dyes with overlapping excitation and/or emission spectra are used together, the spectral overlap between the fluorophores makes it difficult to distinguish one dye from another. be. This is problematic when a panel containing about 5 or more different markers is required to detect a particular disease from a biological specimen, especially when the sample size is small and the markers are present at low levels. In some cases, circumstances often require the use of an entire sample in a single amplification run.

In some embodiments, the methods described herein allow detection of multiple different markers in the same sample by generating results from the same dye in each sample. In embodiments detailed herein, a multiplexed flap cleavage method for multiple different markers (e.g., the QuARTS flap endonuclease method) uses the same FRET cassette to generate multiple initial cleavage products that generate fluorescent signals. be done.

In a preferred embodiment, a multiplex assay comprises several different probe oligonucleotides each having a portion that hybridizes to a different target nucleic acid, but all probe oligonucleotides have essentially identical 5' arm sequences. have. Cleavage of each probe in the presence of its respective target nucleic acid releases the same 5′ arm from all probes, after which all released arms bind to the FRET cassette with the same flap binding sequence and the same dye. and a fluorescent signal is generated by endonuclease cleavage of the FRET cassette. In other embodiments, probes for different targets may have different flap arms and report to different FRET cassettes, where the same reporter fluorophore is used for all such different FRET cassettes.

Combining assays in this manner has a number of advantages. For example, if any target sequence associated with a condition (e.g., a disease state such as colon cancer) is detected in an assay, results can be obtained from one sample without splitting the sample into multiple different assays. can. Additionally, if such results were obtained from more than one of the target sequences, combining these signals into a single dye channel could yield a stronger signal over background, providing additional confidence in the assay results. can be provided. During the development of the methods described herein, it was surprisingly discovered that multiple primers and flap method probes were combined to detect multiple different target sequences, used with shared FRET cassettes, and combined amplification and flap cleavage methods. It was found that the background signal in non-target controls or negative samples was not enhanced when run in a single reaction.

In some embodiments, the different target sequences reporting to a single FRET cassette and single dye channel may not come from different marker genes or regions, but from a single marker (e.g., a single methylation marker gene ) can be derived from different regions within the Configure assays to detect multiple regions of a single marker gene in one assay, all regions reporting to a single dye, e.g., via a single FRET cassette, as described in Example 4. enhances the level of detectable signal from multiple copies of the target gene present in the reaction.

In yet another embodiment, the different target sequences to be detected may be mixtures of one or more regions of different marker(s), as well as multiple regions of one marker. Different target sequences may contain any combination of methylation markers, mutation markers, deletions, insertions, or any other manner of nucleic acid variants detectable in an assay such as the QuARTS amplification/flap cleavage method.

In some embodiments, the marker is a region of 100 bases or less, the marker is a region of 500 bases or less, the marker is a region of 1000 bases or less, or the marker is 5000 bases or less. or, in some embodiments, the marker is a single base. In some embodiments, the marker is within the CpG high density promoter.

Techniques are not limited by sample type. For example, in some embodiments, the sample is a stool sample, tissue sample, sputum, blood sample (eg, plasma, serum, whole blood), stool, or urine sample.

Furthermore, the technique is not limited to the method used to determine methylation status. In some embodiments, assays use methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, chip or array hybridization, methylation-specific nucleases, separation by mass, or target capture. including doing In some embodiments, assays involve the use of methylation-specific oligonucleotides. In some embodiments, techniques include massively parallel sequencing (e.g., next-generation sequencing) to determine methylation status, e.g., synthetic-time decoding, real-time (e.g., single-molecule) sequencing, bead-emulsion sequencing. , using nanopore sequencing, etc.

The technology provides reagents for differentially methylated region (DMR) detection. In some embodiments, oligonucleotides are provided that include sequences complementary to chromosomal regions. ZNF671; CHST2; FLI1; JAM3; SFMBT2; A kit is provided that includes a control nucleic acid that includes a chromosomal region having a methylation status associated with a subject that is not (eg, colon cancer). In some embodiments, the kit includes a bisulfite reagent and an oligonucleotide described herein. In some embodiments, the kit includes a control nucleic acid comprising a sequence derived from such a chromosomal region and having a methylation status associated with a subject with colon cancer, and a bisulfite reagent.

The technology relates to embodiments of compositions (eg, reaction mixtures). FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; A composition is provided. FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; Compositions comprising oligonucleotides are provided. FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; Compositions are provided comprising: FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; provided.

Further embodiments of related methods for screening a sample obtained from a subject for a tumor (e.g., colon carcinoma) are provided, e.g., the methods are VAV3; ZNF671; CHST2; ZNF568; GRIN2D, and QKI. comparing the methylation status of the marker to the control sample; and determining the confidence interval and/or p-value of the difference between the methylation status of the subject sample and the methylation status of the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99% and the p-value is 0 .1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to obtain the nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence to a nucleotide sequence of a nucleic acid comprising a chromosomal region of a subject who does not have colon cancer and identifying differences between the two sequences; if differences exist, identifying such subject as having a tumor. Steps are provided.

A system for screening for colon cancer in a sample obtained from a subject is provided by the present technology. Exemplary embodiments of the system include, for example, a system for screening a sample obtained from a subject for colon cancer, wherein such system comprises an analytical component configured to determine the methylation status of the sample; a software component configured to compare the methylation status of the sample obtained with the methylation status of a control or standard sample recorded in a database; Contains an alert component configured to alert. In some embodiments, the alert is determined by a software component that receives results from multiple assays (e.g., multiple markers, e.g., VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and determining the methylation status of chromosomal regions with annotations selected from QKI, etc., calculating values or results and reporting on multiple results). ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; A database of weighting parameters associated with each chromosomal region with annotations selected from and/or alerts reporting to users (eg, doctors, nurses, clinicians, etc.) is provided. In some embodiments, the total results of multiple assays are reported, and in some embodiments, one or more results are used to combine the results of the subject's colon based on a composite of one or more results from multiple assays. A score, value or outcome indicative of cancer risk is provided.

FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; . In some embodiments, the system further includes a sample collection component, such as a nucleic acid isolation component, a stool sample collection component, and the like. FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; In some embodiments, the database contains nucleic acid sequences of subjects who do not have colon cancer. FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; provided.

Embodiments of related systems include the described nucleic acid sets and databases of nucleic acid sequences associated with such nucleic acid sets. Some embodiments further include a bisulfite reagent. Some embodiments also include a nucleic acid sequencer.

In certain embodiments, a method of characterizing a sample obtained from a human subject is provided, comprising: a) obtaining the sample from the human subject; b) determining the methylation status of one or more markers in the sample; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; c) comparing the methylation status of the markers assayed with the methylation status of the markers assayed in tumor-free subjects.

In some embodiments, the techniques involve assessing the presence and methylation status of one or more of the markers identified herein in a biological sample. These markers include one or more of the differentially methylated regions (DMRs) discussed herein. Methylation status is assessed in embodiments of the technology. Therefore, the techniques provided herein are not bound by methods of measuring the methylation status of genes. For example, in some embodiments, methylation status is measured by genome scanning methods. For example, one method involves genome scanning with landmark restriction enzyme cleavage sites (Kawai et al. (1994) Mol. Cell. Biol. 14:7421-7427); Primed PCR (Gonzalgo et al. (1997) Cancer Res. 57:594-599) is performed. In some embodiments, changes in methylation patterns at specific CpG sites are monitored by digesting genomic DNA with methylation-sensitive restriction enzymes followed by Southern analysis of the region of interest (digestion-Southern method). do. In some embodiments, analysis of changes in methylation patterns uses PCR-based methods in which genomic DNA is digested with methylation-sensitive restriction enzymes prior to PCR amplification (Singer-Sam et al. (1990). ) Nucl.Acids Res. 18:687). In addition, other techniques have been reported that utilize bisulfite treatment of DNA as a starting point for methylation analysis. These include methylation-specific PCR (MSP) (Herman et al. (1992) Proc. Natl. Acad. Sci. USA 93:9821-9826) and PCR products amplified from bisulfite-converted DNA. Includes restriction enzyme digestion (Sadri and Hornsby (1996) Nucl. Acids Res. 24:5058-5059; and Xiong and Laird (1997) Nucl. Acids Res. 25:2532-2534). PCR methods for detecting genetic mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147) and PCR methods for quantifying allele-specific expression (Szabo and Mann 1995) Genes Dev.9:3097-3108; and Singer-Sam et al.(1992) PCR Methods Appl.1:160-163). Such techniques employ internal primers that anneal with a PCR-generated template to form a direct terminus 5' to the single nucleotide to be assayed. Some embodiments utilize a method using the “quantitative Ms-SNuPE method” described in US Pat. No. 7,037,650.

When assessing methylation status, methylation status may be determined at specific sites (e.g., single nucleotides, specific regions or loci, longer sequences of interest, e.g., up to about 100 bp, 200 bp, 500 bp, 1000 bp, or more). It is often expressed as the amount or percentage of an individual strand of DNA that is methylated at that particular site relative to the total DNA population in a sample, including DNA subsequences). Conventionally, the amount of unmethylated nucleic acid is determined by PCR using calibrators. Known amounts of DNA are then bisulfite treated and the resulting methylation-specific sequences are subjected to real-time PCR or other exponential amplification methods, such as the QuARTS method (e.g., US Pat. No. 8,361,720; Nos. 8,715,937, 8,916,344, and 9,212,392).

For example, in some embodiments, the method includes generating a standard curve of unmethylated targets using an external standard. A standard curve is generated from at least two points, relating real-time Ct values of unmethylated DNA to known quantitation standards. A second standard curve of methylated targets is then generated from the at least two points and an external standard. This second standard curve relates the Ct of methylated DNA to known quantitation standards. Ct values of test samples are then determined in methylated and unmethylated populations, and genome equivalents of DNA are calculated from the standard curves obtained in the first two steps. The methylation ratio at the site of interest is calculated from the amount of methylated DNA relative to the total amount of DNA in the population, for example, (number of methylated DNA)/(number of methylated DNA+number of unmethylated DNA)×100.

Also provided herein are compositions and kits for carrying out the methods. For example, in some embodiments, reagents (eg, primers, probes) specific for one or more markers are provided singly or in sets (eg, sets of primer pairs that amplify multiple markers). Additional reagents for performing detection methods may also be provided (eg, positive and negative controls, enzymes, buffers, etc. for performing QuARTS methods, PCR methods, sequencing, bisulfite methods, or other assays). In some embodiments, kits are provided that contain one or more reagents necessary, sufficient, or useful for the practice of the methods. Also provided is a reaction mixture containing the reagents. A master mix reagent set containing a plurality of reagents is further provided, which may each be added to each other and/or to the test sample to form a complete reaction mixture.

DNA isolation methods suitable for these assay techniques are known in the art. In particular, some embodiments involve isolation of nucleic acids as described in US Pat. No. 9,000,146, which is incorporated herein by reference in its entirety.

Genomic DNA can be isolated by any means, such as using commercially available kits. Briefly, if the DNA of interest is encapsulated by cell membranes, the biological sample must be disrupted and lysed by enzymatic, chemical or mechanical means. Proteins and other contaminants may then be removed from the DNA solution, such as by digestion with proteinase K, for example. The genomic DNA is then recovered from the solution. This can be done by means of a wide variety of methods, such as salting out, organic extraction, or DNA binding to a solid support. The choice of method is influenced by several factors such as time, cost, and amount of DNA required. Any type of clinical sample containing neoplastic or preneoplastic material is suitable for use in the methods of the present application, e.g., cell lines, histological slides, biopsies, paraffin-embedded tissue, body fluids, faeces, colonic effluent Fluid, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof.

The technique is not limited to the method used to prepare the sample and obtain the nucleic acid for testing. For example, in some embodiments, DNA is captured using direct gene capture, such as the methods detailed in US Pat. Nos. 8,808,990 or 9,000,146, or related methods. Isolate from fecal samples, or from blood, or from plasma samples.

The technology relates to analysis of any sample related to colon cancer. For example, in some embodiments the sample comprises tissue and/or biological fluid obtained from a patient. In some embodiments the sample comprises a secretion. In some embodiments, the sample comprises colon DNA recovered from sputum, blood, serum, plasma, gastric secretions, colon tissue samples, colon cells or feces. In some embodiments, the subject is human. Such samples can be obtained by any number of means known in the art and will be apparent to those skilled in the art.

[I. Methylation Assay to Detect Colon Cancer]
Selected colon cancer case and colon control tissues were subjected to unbiased whole-methylome sequencing to identify candidate methylated DNA markers. Top marker candidates were further evaluated in 89 cancer and 95 normal plasma samples. DNA extracted from patient tissue samples is treated with bisulfite, followed by standard genes (eg, β-actin or B3GALT6) as normalization genes and candidate markers in a quantitative allele-specific real-time target and signal amplification method. (Quantitative Allele-Specific Real-time Target and Signal amplification) (QuARTS amplification method). The QuARTS method chemistry is highly discriminating in the selection and screening of methylation markers.

The area under the curve (AUC) ranged from 0.63 to 0.75 in the receiver operator characterization of individual marker candidates. A combined panel of 12 methylation markers (VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; combination resulted in a sensitivity of 67.4% across all stages of colon cancer.

[II. Methylation detection method and kit]
The markers described herein find use in a wide variety of methylation detection methods. The method most frequently used for nucleic acid analysis for the presence of 5-methylcytosine is the bisulfite method for detecting 5-methylcytosine in DNA, described by Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-31) or variations thereof, which are expressly incorporated herein in their entirety. The bisulfite method of mapping 5-methylcytosine is based on the observation that cytosine reacts with bisulfite ions (also known as bisulfite), but 5-methylcytosine does not. The reaction is typically carried out according to the following steps. First, cytosine is reacted with bisulfite to form sulfonated cytosine. The sulfonated reaction intermediate then spontaneously deaminates to sulfonated uracil. Finally, under alkaline conditions, the sulfonated uracil is desulfonated to produce uracil. Uracil base pairs with adenine (thus behaves like thymine) and 5-methylcytosine base pairs with guanine (thus behaves like cytosine) and is detectable. This allows discrimination between methylated and unmethylated cytosines, for example in bisulfite genome sequencing (Grigg G, & Clark S, Bioessays (1994) 16:431-36; Grigg G, DNA Seq. (1996)). 6:189-98), methylation-specific PCR (MSP), such as those disclosed in US Pat. No. 5,786,146, or assays involving sequence-specific probe cleavage, such as the QuARTS flap endonuclease. (e.g., Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology” Clin Chem 56:A199; and U.S. Patent No. 8,361,720 No. 8,715,937, No. 8,916,344 and 9,212,392).

Some conventional techniques include encapsulating the DNA to be analyzed in an agarose matrix to prevent diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and rapid dialysis for precipitation and purification steps. (Olek A, et al. (1996) "A modified and improved method for bisulfite based cytosine methylation analysis" Nucleic Acids Res. 24:5064-6). It is thus possible to analyze individual cells for methylation status, demonstrating the utility and sensitivity of the method. A review of conventional methods for detecting 5-methylcytosine can be found in Rein, T.; , et al. (1998) Nucleic Acids Res. 26:2255.

Bisulfite methods typically involve bisulfite treatment followed by amplification of short specific fragments of known nucleic acids, followed by assay of the products by sequencing (Olek & Walter (1997) Nat. Genet. 17:275- 6), or by a primer extension reaction (Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-31; WO95/00669; US Pat. No. 6,251,594) to analyze the position of individual cytosines. Other methods utilize enzymatic digestion (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-4). Detection methods by hybridization are also described in the art (Olek et al., WO99/28498). Additionally, the use of the bisulfite method to detect methylation for individual genes has been described (Grigg & Clark (1994) Bioessays 16:431-6; Zeschnigk et al. (1997) Hum Mol Genet. 6: 387-95; Feil et al.(1994) Nucleic Acids Res.22:695; Martin et al.(1995) Gene 157:261-4;

Various methylation assay procedures can be performed in conjunction with bisulfite treatment according to the present technology. These assays allow measurement of the methylation status of one or more CpG dinucleotides (eg, CpG islands) within a nucleic acid sequence. Such assays involve, among other techniques, sequencing of bisulfite-treated nucleic acids, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-sensitive restriction enzymes.

For example, genomic sequencing to analyze the methylation pattern and distribution of 5-methylcytosine by using bisulfite treatment (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-1831). Thing is simplified. Additionally, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is described, for example, in Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058-5059, or exemplified in a method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534). Used for evaluation of methylation status.

The COBRA™ method is a quantitative methylation assay useful for determining DNA methylation levels at specific loci with small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. Methylation-dependent sequence differences are first introduced into genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfite-converted DNA is then performed using primers specific for the CpG island of interest, followed by restriction endonuclease digestion, gel electrophoresis, and specific label hybridization. Perform detection using probes. The methylation level of the original DNA sample is expressed in a quantitative linear fashion over a wide range of DNA methylation levels by the relative amount of digested and undigested PCR products. Moreover, this technique is reliably applicable to DNA obtained from microdissected paraffin-embedded tissue samples.

Exemplary reagents for COBRA™ analysis (e.g., those included in kits using exemplary COBRA™) include specific loci (e.g., specific genes, markers, regions of genes). , regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.); restriction enzymes and appropriate buffers; gene hybridization oligonucleotides; control hybridization oligonucleotides; Examples include, but are not limited to, labeled nucleotides. Additionally, bisulfite conversion reagents include DNA denaturation buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; elements can be included.

"MethyLight™" (real-time PCR method by fluorescence) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE™ (Methylation-sensitive Single Nucleotide Primer Extension) single nucleotide primer extension) reaction (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR ("MSP"; Herman et al., Proc. Natl. Acad. Sci. USA 93:9821- 9826, 1996; US Pat. No. 5,786,146), and methylated CpG island amplification (“MCA”; Toyota et al., Cancer Res. 59:2307-12, 1999). or in combination with one or more of these methods.

The "HeavyMethyl™" method technique is a quantitative method for assessing methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes (“blockers”) that encompass CpG positions flanked by or occupied by amplification primers allow methylation-specific selective amplification of a nucleic acid sample.

The term "HeavyMethyl™ MethyLight™" method refers to the HeavyMethyl™ MethyLight™ method, which is a variant of the MethyLight™ method and includes methylation-specific CpG positions flanked by amplification primers. It combines a functional blocking probe with the MethyLight™ method. The HeavyMethyl™ method may be used in conjunction with methylation-specific amplification primers.

Exemplary reagents for HeavyMethyl analysis (e.g., reagents such as those in kits using exemplary MethyLight™) include specific loci (e.g., specific genes, markers, regions of genes, markers of regions, bisulfite-treated DNA sequences, CpG islands, or bisulfite-treated DNA sequences or CpG islands); blocking oligonucleotides; optimized PCR buffers and deoxynucleotides; and Taq polymerase. but not limited to this.

MSP (methylation-specific PCR) allows the assessment of methylation status at virtually any group of CpG sites within a CpG island, with or without the use of methylation-sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; Briefly, DNA is modified with sodium bisulfite, which converts unmethylated cytosines to uracil but not methylated cytosines to uracil, and then converts the products to specific methylated DNA. Amplification is performed using specific primers and primers specific for unmethylated DNA. MSP requires only small amounts of DNA, is sensitive to 0.1% methylated alleles at a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Exemplary reagents for MSP analysis (e.g., reagents such as those included in kits using exemplary MSPs) include specific loci (e.g., specific genes, markers, regions of genes, regions of markers, overlapping both methylated and unmethylated PCR primers for sulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides, and specific probes, including but not limited to do not have.

The MethyLight™ method is a high-throughput, quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) without the need for further manipulation after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™ process starts with a mixed sample of genomic DNA and converts it in a sodium bisulfite reaction according to standard procedures into a mixed pool with methylation-dependent sequence differences (multiple A sulfite-based step converts unmethylated cytosine residues to uracil). Fluorescent PCR is then performed, eg, in a “biased” reaction with PCR primers overlapping known CpG dinucleotides. Sequence discrimination is performed at both the amplification step level and the fluorescence detection step level.

The MethyLight™ method is used as a quantitative test of methylation patterns of nucleic acids, such as genomic DNA samples, where sequence discrimination is performed at the probe hybridization level. In the quantitative version, the PCR reaction results in methylation-specific amplification in the presence of fluorescent probes overlapping specific putative methylation sites. An unbiased control for the amount of input DNA is obtained with reactions in which neither the primer nor the probe covers the CpG dinucleotides. Alternatively, qualitative testing of genomic methylation can be performed using control oligonucleotides that do not encompass known methylation sites (e.g., HeavyMethyl™ and fluorescent versions of the MSP technique), or This is accomplished by probing a biased PCR pool with oligonucleotides encompassing the site.

The MethyLight™ process is used with appropriate probes (eg, “TaqMan®” probes, Lightcycler® probes, etc.). For example, in some applications double-stranded genomic DNA is treated with sodium bisulfite and two sets of PCR reactions using TaqMan® probes, e.g., MSP primers and/or HeavyMethyl blocker oligonucleotides and TaqMan® ) are subjected to one set of PCR reactions using the probes. TaqMan® probes are dual-labeled with fluorescent “reporter” and “quencher” molecules and are GC-enhanced so that they melt approximately 10° C. higher than the forward or reverse primers in the PCR cycle. Designed to be specific to regions of relatively high content. This allows the TaqMan® probes to remain fully hybridized during the annealing/extension steps of PCR. During PCR, Taq polymerase enzymatically synthesizes new strands, eventually reaching the annealed TaqMan® probe. The 5′ to 3′ endonuclease activity of Taq polymerase then digests and displaces the TaqMan® probe, liberating the fluorescent reporter molecule and dequenching its signal using a real-time fluorescence detection system. Quantitative detection using

Exemplary reagents for MethyLight analysis (e.g., those included in kits using exemplary MethyLight™) include specific loci (e.g., specific genes, markers, regions of genes, markers of regions, bisulfite-treated DNA sequences, CpG islands, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides; It is not limited to this.

The QM™ (quantitative methylation) method is an alternative quantitative test for methylation patterns in genomic DNA samples, in which sequence discrimination is performed at the probe hybridization level. In this quantitative version, PCR reactions yield unbiased amplification in the presence of fluorescent probes that overlap with specific putative methylation sites. An unbiased control for the amount of input DNA is obtained with reactions in which neither the primer nor the probe covers the CpG dinucleotides. Alternatively, qualitative testing of genomic methylation can be performed using control oligonucleotides that do not encompass known methylation sites (e.g., HeavyMethyl™ and fluorescent versions of the MSP technique), or This is accomplished by probing a biased PCR pool with oligonucleotides encompassing the site.

The QM™ process can be used in the amplification step using appropriate probes, eg, "TaqMan®" probes, Lightcycler® probes. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and TaqMan® probes. TaqMan® probes are dual-labeled with fluorescent “reporter” and “quencher” molecules and are GC-enhanced so that they melt approximately 10° C. higher than the forward or reverse primers in the PCR cycle. Designed to be specific to regions of relatively high content. This allows the TaqMan® probes to remain fully hybridized during the annealing/extension steps of PCR. During PCR, Taq polymerase enzymatically synthesizes new strands, eventually reaching the annealed TaqMan® probe. The 5′ to 3′ endonuclease activity of Taq polymerase then digests and displaces the TaqMan® probe, liberating the fluorescent reporter molecule and dequenching its signal using a real-time fluorescence detection system. Quantitative detection using Exemplary reagents for QM™ analysis (e.g., reagents such as those included in kits using exemplary QM™) include specific loci (e.g., specific genes, markers, regions of genes). , regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides; but not limited to this.

The Ms-SNuPE™ method is a quantitative method to assess methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by single-nucleotide primer extension (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosines to uracils, leaving 5-methylcytosines unchanged. PCR primers specific for the bisulfite-converted DNA are then used to amplify the desired target sequence and the resulting product is isolated and used as a template for methylation analysis at the CpG site of interest. do. The ability to analyze small amounts of DNA (eg, microdissected pathology sections) avoids the use of restriction enzymes to determine methylation status at CpG sites.

Exemplary reagents for Ms-SNuPE™ analysis (eg, reagents such as those in kits using exemplary Ms-SNuPE™) include specific loci (eg, specific genes, markers , regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides; gel extraction kits; positive control primers; -SNuPE™ primers; reaction buffer (for Ms-SNuPE reactions); and labeled nucleotides, including but not limited to. Additionally, bisulfite conversion reagents include DNA denaturation buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; elements can be included.

Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines to uracils, followed by restriction enzymes (such as MspI). , an enzyme that recognizes sites containing CG sequences), and the fragments are bound to adapter ligands before complete sequencing. The choice of restriction enzymes results in more CpG-dense regions in the fragment and reduces the number of redundant sequences that can be located at multiple gene positions during analysis. Thus, RRBS reduces the complexity of nucleic acid samples by selecting a subset of restriction fragments for sequencing (eg, by size selection using preparative gel electrophoresis). In contrast to whole-genome bisulfite sequencing, any fragment generated by restriction enzyme digestion contains DNA methylation information for at least one CpG dinucleotide. Thus, in RRBS, samples are enriched for promoters, CpG islands, and other features of the genome, and are associated with a high frequency of restriction enzyme cleavage sites within these regions, resulting in one or more genomic loci. Assays to assess methylation status are provided.

A typical protocol for RRBS involves digesting a nucleic acid sample with a restriction enzyme such as MspI, filling in protruding ends and adding A tails, ligating adapters, converting with bisulfite, and performing PCR. . For example, et al. (2005) "Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution" Nat Methods 7:133-6; Meissner et al. (2005) "Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis" Nucleic Acids Res. 33:5868-77.

In some embodiments, the quantitative allele-specific real-time target and signal amplification (QuARTS) method is used to assess methylation status. Three reactions were performed sequentially for each QuARTS method, including amplification (reaction 1) and target probe cleavage (reaction 2) in the primary reaction; and FRET cleavage and fluorescence signal generation (reaction 3) in the secondary reaction. included. Certain detector probes with flap sequences bind loosely to amplicons when using certain primers to amplify a target nucleic acid. Upon the presence of a specific invasion oligonucleotide at the target binding site, a 5' nuclease, eg, FEN-1 endonuclease, cleaves between the detection probe and the flap sequence liberating the flap sequence. The flap sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. Thus, the flap sequence functions as an invasion oligonucleotide on the FRET cassette, performing cleavage between the FRET cassette's fluorophore and quencher, thereby generating a fluorescent signal. The cleavage reaction can cleave multiple probes per target, thereby liberating multiple fluorophores per flap, resulting in exponential signal amplification. QuARTS allows detection of multiple targets in a single reaction well through the use of different dye FRET cassettes. (See, eg, Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology” Clin Chem 56:A199). In embodiments described herein, the QuARTS method is used to detect multiple different targets or different regions of the same target using the same FRET cassette to generate an additive fluorescent signal from a single dye. Can also be configured.

In some embodiments, the bisulfite-treated DNA is purified prior to quantification. This purification may be performed by any means known in the art such as, but not limited to, ultrafiltration, for example Microcon™ columns (manufactured by Millipore™). Purification is performed according to a modified manufacturer's protocol (see, eg, PCT/EP2004/011715, which is incorporated by reference in its entirety). In some embodiments, the bisulfite-treated DNA is bound to a solid support, such as magnetic beads, and desulfonated and washed while the DNA remains bound to the solid support. Examples of such embodiments are described, for example, in WO2013/116375. In certain preferred embodiments, the support-bound DNA is ready for methylation assays immediately after desulfonation and washing on the support. In some embodiments, the desulfonated DNA is eluted from the support prior to performing the assay.

In some embodiments, fragments of treated DNA are amplified using a primer oligonucleotide set according to the invention (see, eg, FIG. 1) and amplification enzymes. Amplification of several DNA segments can be performed simultaneously in one and the same reaction vessel. Amplification is typically performed using the polymerase chain reaction (PCR).

DNA isolation methods suitable for these assay techniques are known in the art. In particular, some embodiments are described in U.S. Patent Application No. 13/470,251 ("Isolation of Nucleic Acids", published as US2012/0288868), which is incorporated herein by reference in its entirety. including nucleic acid isolation.

In some embodiments, the markers described herein are utilized in the QUARTS method performed on stool samples. In some embodiments, methods of making DNA samples, particularly containing small amounts of highly purified nucleic acids in small volumes (e.g., less than 100 microliters, less than 60 microliters), for use in testing DNA samples Methods are provided for making DNA samples that are substantially and/or virtually free of substances that interfere with an assay (eg, PCR, INVADER method, QuARTS method, etc.). Such DNA samples can be used to qualitatively determine the presence, expression, or amount of genes, genetic variants (e.g., alleles), or genetic modifications (e.g., methylation) present in a sample taken from a patient. It is used in diagnostic tests to detect or measure quantitatively. For example, some cancers are correlated with the presence of certain mutated alleles or methylation states, and thus detection and/or quantification of such mutated alleles or methylation states may be useful in cancers. It has predictive value in diagnosis and therapy.

Many important genetic markers are present in very low amounts in samples, and many of the events in which such markers occur are rare. As a result, even sensitive detection methods such as PCR require large amounts of DNA to obtain sufficient amounts of low-abundance target to meet or exceed the detection threshold of the assay. Furthermore, the presence of inhibitors, even in small amounts, compromises the accuracy and precision of these assays intended for detection of such low-abundance targets. Accordingly, the present specification provides methods for controlling the volumes and concentrations required to generate such DNA samples.

In some embodiments, the sample comprises blood, serum, plasma, or saliva. In some embodiments, the subject is human. Such samples can be obtained by any number of means known in the art and will be apparent to those skilled in the art. A cell-free or substantially cell-free sample can be obtained by subjecting the sample to a wide variety of techniques known to those of skill in the art, including, but not limited to, centrifugation and filtration. Although it is generally preferred to obtain samples without using invasive techniques, it may nevertheless be preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. The technique is not limited to the method used to prepare the sample and obtain the nucleic acid for testing. For example, in some embodiments, direct gene capture or related methods, such as those detailed in U.S. Pat. is used to isolate DNA from stool samples or blood or plasma samples.

Analysis of markers can be performed separately or simultaneously with additional markers within one test sample. For example, several markers can be combined into one test to efficiently process multiple samples and to be able to provide high diagnostic and/or prognostic accuracy. Furthermore, those skilled in the art will recognize the value of testing multiple samples of the same subject (eg, at consecutive time points). Examination of such serially collected samples can identify changes in the methylation status of markers over time. Changes in methylation status, as well as absence of change in methylation status, can provide useful information about disease status, including determination of approximate time from event onset, remedial This includes, but is not limited to, confirmation of a subject's outcome, including the presence and amount of healthy tissue, adequacy of drug therapy, efficacy of various treatments, and risk of future events.

Analysis of biomarkers can be done in a variety of physical formats. For example, the use of microtiter plates or automation can be utilized to facilitate processing of large numbers of test samples. Alternatively, a single sample format could be produced to facilitate timely and rapid treatment and diagnosis, eg, in ambulatory transport or emergency room settings.

Embodiments of the technology are intended to be provided in kit form. Kits include embodiments of the compositions, devices, instrumentation, etc. described herein and instructions for use of the kit. Such instructions describe suitable methods for preparing the analyte from the sample, eg, for obtaining the sample and preparing nucleic acids from the sample. The individual components of the kit are packaged in suitable containers and packaging materials (e.g., vials, boxes, blister packs, ampoules, jars, bottles, tubes, etc.), and such components are easy to store, transport, and/or All are packaged together in a suitable container (eg, box(es)) for convenient use by the user of the kit. It is understood that the liquid components (eg, buffers) may be provided in lyophilized form for reconstitution by the user. Kits may include controls or standards to evaluate, confirm, and/or ensure the performance of the kit. For example, a kit for quantifying the amount of nucleic acid contained in a sample can include a control containing a known concentration of the same or another nucleic acid for comparison; detection reagents (eg, primers) may be included. The kits are suitable for use in a clinical setting and, in some embodiments, for use at the user's home. In some embodiments, the components of the kit provide the functionality of the system for preparing nucleic acid solutions from samples. In some embodiments, certain components of the system are provided by the user.

[III. Application]
In some embodiments, a diagnostic test identifies the presence of a disease or condition in an individual. In some embodiments, the disease is cancer (eg, colon cancer). In some embodiments, a marker whose aberrant methylation is associated with colon cancer (e.g., one or more markers selected from those listed in Table 1, or preferably VAV3; ZNF671; CHST2 PDGFD; DTX1; TSPYL5; ZNF568; one or more of GRIN2D, QKI, FER1L4). In some embodiments, the assay further includes detection of canonical genes (eg, β-actin, ZDHHC1, B3GALT6).

In some embodiments, the techniques are applied to treat a patient (e.g., a colon cancer patient, an early colon cancer patient, or a patient at risk of developing colon cancer), the method comprising one of the methods provided herein. Determining the methylation status of the above markers and administering a treatment to the patient based on the results of the previously determined methylation status. The treatment may be administering a pharmaceutical compound, administering a vaccine, performing surgery, imaging the patient, or performing other tests. Preferably, such uses are methods of clinical screening, methods of assessing prognosis, methods of monitoring therapeutic outcome, methods of identifying patients likely to respond to a particular therapeutic treatment, patients or in methods of imaging subjects and methods of drug screening and development.

In some embodiments, the technology is applied to methods of diagnosing colon cancer in a subject. As used herein, the terms "diagnose" and "diagnosis" refer to whether a subject has a given disease or condition or is likely to develop a given disease or condition in the future. can be estimated and further determined by those skilled in the art. Those skilled in the art often base their diagnosis on one or more diagnostic indicators, such as biomarkers, whose methylation status indicates the presence, severity, or absence of a disease state.

Along with diagnosis, clinical prognosis of cancer involves determining the aggressiveness of the cancer and the likelihood of tumor recurrence, and planning the most effective therapy. If a more accurate prognosis can be made, and the potential risk of developing cancer can be assessed, appropriate treatment, possibly less severe treatment for the patient, can be selected. Evaluation of cancer biomarkers (e.g., determination of methylation status) should be more intensive in subjects with favorable prognosis and/or low risk of developing cancer who do not require or require limited treatment. It is useful in distinguishing subjects likely to develop cancer or suffering from cancer recurrence who would benefit from treatment.

Thus, as used herein, "making a diagnosis" or "diagnosing" further includes making a risk assessment for developing cancer or determining a prognosis, thereby Predicting clinical outcome (with or without drug therapy), selecting appropriate treatment (or whether treatment is effective), or monitoring current treatment based on the measurement of diagnostic biomarkers disclosed herein and possible therapeutic modifications can be provided.

Further, in some embodiments of the technology, multiple determinations of biomarkers can be made over time to facilitate diagnosis and/or prognosis. Changes in biomarkers over time can be used to predict clinical outcome, monitor colon cancer progression, and/or monitor the effectiveness of appropriate therapies for cancer. For example, in such embodiments, one or more biomarkers disclosed herein (and possibly one or more additional biomarker(s) if monitored) in a biological sample during effective therapy ) would be expected to change over time.

The technology is further applied to methods for deciding on initiation or continuation of cancer prevention or treatment in a subject. In some embodiments, the method comprises providing a series of biological samples from a subject over time; analyzing such series of biological samples to determine the methylation status of at least one biomarker disclosed herein in vivo. determining in each of the samples; and comparing a measurable change in the methylation status of one or more biomarkers in each of the biological samples. Using changes in the methylation status of biomarkers over time to predict the risk of developing cancer, predict clinical outcome, make decisions about initiation or continuation of cancer prevention or treatment, and current can determine whether cancer is effectively treated by treatment of For example, a first time point may be selected prior to initiation of treatment and a second time point may be selected at some time after initiation of treatment. The methylation status can be measured in each of the samples taken from different time points and any qualitative and/or quantitative differences can be described. Changes in biomarker-level methylation status obtained from different samples can be correlated with the risk of developing colon cancer, prognosis, treatment efficacy, and/or cancer progression in a subject.

In preferred embodiments, the methods and compositions of the invention are for treating or diagnosing disease early, eg, before symptoms of the disease appear. In some embodiments, the methods and compositions of the invention are for treating or diagnosing disease at the clinical stage.

As noted above, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and changes in the markers over time are used to determine diagnosis or prognosis. can be done. For example, diagnostic markers can be determined a first time and again a second time. In such embodiments, a first to second increase in a marker can lead to diagnosis of a particular type or severity of cancer, or a given prognosis. Similarly, a decrease in a marker from the first to the second may indicate a particular type or severity of cancer, or a given prognosis. In addition, the degree of change in one or more markers can be related to cancer severity and future adverse events. One skilled in the art will measure a given biomarker at one time point and a second biomarker at a second time point, although in some embodiments comparative measurements can be made for the same biomarker at multiple time points. It will be appreciated that comparison of these markers can provide diagnostic information.

As used herein, the phrase "determine prognosis" refers to methods by which the skilled artisan can predict the course or outcome of a condition in a subject. The term "prognosis" does not refer to the ability to predict the course or outcome of a disease state with 100% accuracy, nor does it predict the likelihood of a given course or outcome based on the methylation status of biomarkers. It doesn't mean you can do it. Instead, the term "prognosis" refers to the likelihood that a particular course or outcome will occur, i.e., a subject exhibiting a given condition is more likely to have a certain course or outcome than an individual not exhibiting such condition. It can be understood to refer to the likelihood that an outcome will occur. For example, an individual who does not exhibit such a condition may be very unlikely to have a given outcome (eg, develop colon cancer).

In some embodiments, statistical analysis associates prognostic indicators with predispositions for adverse outcomes. For example, in some embodiments, a methylation state that differs from that of a normal control sample obtained from a non-cancer patient, as determined at the level of statistical significance, is the It may suggest that a subject with a similar level of inflammatory status is more likely to have cancer than a subject. Further, changes in methylation status from baseline (eg, "normal") levels can reflect the prognosis of a subject, and the degree of change in methylation status can be related to severity of adverse events. Statistical significance is often determined by comparing two or more populations and determining confidence intervals and/or p-values. See, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, which is hereby incorporated by reference in its entirety. Exemplary confidence intervals for the subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, and exemplary p-values are , 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

In other embodiments, a threshold for the degree of change in the methylation status of a prognostic or diagnostic biomarker disclosed herein can be set, and the degree of change in the methylation status of a biomarker in a biological sample is is simply compared with a threshold for the degree of change in methylation status. Preferred thresholds for changes in methylation status of biomarkers provided herein are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75% , about 100%, and about 150%. In yet another embodiment, a "nomogram" can be established whereby the methylation status of a prognostic or diagnostic indicator (one or a combination of biomarkers) is associated with a given outcome. is directly related to the predisposition to Those skilled in the art are familiar with the use of nomograms that relate two such numbers, and because they refer to individual sample measurements rather than population averages, the uncertainty in this measurement is the marker concentration We understand that it is the same as uncertainty in

In some embodiments, a control sample is analyzed at the same time as the biological sample so that the results obtained from the biological sample can be compared to the results obtained from the control sample. Further, it is contemplated that a standard curve may be provided by which assay results of biological samples may be compared. Such a standard curve represents the methylation status of the biomarkers as a function of assay units, eg, fluorescence signal intensity when fluorescent labels are used. Using samples from multiple donors, a control that is the methylation status of one or more biomarkers in normal tissue, as well as a "risk" of one or more biomarkers in tissue from colon cancer donors A calibration curve can be obtained for the levels.

Analysis of markers can be performed separately or simultaneously with additional markers within one test sample. For example, several markers can be combined into one test to efficiently process multiple samples and to be able to provide high diagnostic and/or prognostic accuracy. Furthermore, those skilled in the art will recognize the value of testing multiple samples of the same subject (eg, at consecutive time points). Examination of such serially collected samples can identify changes in the methylation status of markers over time. Changes in methylation status, as well as absence of change in methylation status, can provide useful information about disease status, including determination of approximate time from event onset, remedial This includes, but is not limited to, confirmation of a subject's outcome, including the presence and amount of healthy tissue, adequacy of drug therapy, efficacy of various treatments, and risk of future events.

Analysis of biomarkers can be done in a variety of physical formats. For example, the use of microtiter plates or automation can be utilized to facilitate processing of large numbers of test samples. Alternatively, a single sample format could be created to facilitate timely and rapid treatment and diagnosis, eg, in ambulatory transport or emergency room settings.

In some embodiments, a subject is diagnosed with colon cancer if there is a measurable difference in the methylation status of at least one biomarker in the sample when compared to the methylation status of the control. Conversely, if no altered methylation status is identified in the biological sample, the subject is identified as not having colon cancer, not at risk for colon cancer, or at low risk for colon cancer. In this regard, one can distinguish between subjects who have or are at risk of colon cancer and those who are at or at low or substantially no risk of colon cancer. Subjects at risk of developing colon cancer can undergo a more intensive and/or regular screening schedule. On the other hand, low risk and substantially no risk subjects are subjected to screening methods until subsequent screening, such as screening performed in accordance with the present technology, indicates that they are at risk for colon cancer. Sometimes you don't have to.

As noted above, depending on the embodiment of the methods of the present technology, detecting a change in methylation status of one or more biomarkers can be a qualitative or quantitative determination. Thus, diagnosing a subject as having or being at risk of developing colon cancer indicates that a particular threshold measurement has been made, e.g. The methylation state is different from a given control methylation state. In some embodiments of the method, the control methylation status is any detectable methylation status of the biomarker. In other embodiments of the method in which the control sample and the biological sample are tested simultaneously, the predetermined methylation state is that of the control sample. In other embodiments of the method, the predetermined methylation state is based on and/or specified by a standard curve. In other embodiments of the method, the predetermined methylation state is a particular state or a particular range of states. Thus, a given methylation state can be chosen within acceptable limits that will be apparent to those skilled in the art, based in part on the embodiment of the method being practiced, the specificity desired, and the like.

In recent years it has become clear that circulating epithelial cells representing metastatic tumor cells can be detected in the blood of many cancer patients. Molecular profiling of rare cells is important in biological and clinical research. Applications are available from the characterization of cancer patient peripheral blood circulating epithelial cells (CEpC) for disease prognosis and personalized medicine (e.g. Cristofanilli M, et al. (2004) N Engl J Med 351: Hayes DF, et al.(2006) Clin Cancer Res 12:4218-4224;Budd GT, et al.,(2006) Clin Cancer Res 12:6403-6409;Moreno JG, et al.(2005) Urology 65:713-718; Pantel et al., (2008) Nat Rev 8:329-340; and Cohen SJ, et al. (2008) J Clin Oncol 26:3213-3221). Accordingly, embodiments of the present disclosure provide compositions and methods for detecting the presence of metastatic cancer in a subject by ascertaining the presence of methylation markers in the subject's plasma or whole blood.
[Example]

[Sample preparation method]
[DNA isolation method and QUARTS method]
The following provides an exemplary pre-analytical DNA isolation method, and an exemplary QuARTS method, as may be used in accordance with embodiments of the technology. Although this example describes the application of the QuARTS method to DNA from blood and various tissue samples, such techniques are readily applied to other nucleic acid samples, as shown in other examples.

[DNA isolation from cells and plasma]
For cell lines, for example, the Maxwell® RSC ccfDNA Plasma Kit (Promega Corp., Madison, Wis.) may be used to isolate genomic DNA from cell-conditioned media. Conditioned medium (CCM) is used in place of plasma and processed according to the kit procedure.The elution volume is 100 μL, of which typically 70 μL is used for bisulfite conversion, each of which is referenced for all purposes. U.S. Patent Application Nos. 62/249,097, filed Oct. 30, 2015; and 15/335,096; and International Application No. PCT/US16/58875, filed October 26, 2016.

An example of the overall process for isolating DNA from a blood sample, eg, for use in detection methods, is provided in this example. Optional bisulfite conversion and detection methods are also described.

[I. Blood processing]
Collect whole blood into tubes with anticoagulant EDTA or Streck Cell-Free DNA BCT. An exemplary procedure is as follows.

1. Collect 10 mL of whole blood into a vacutainer tube (anticoagulant EDTA or Streck BCT) and collect the maximum volume to ensure the correct ratio of blood to anticoagulant.

2. After collection, gently mix the blood by inverting the tube 8-10 times to mix the blood and anticoagulant, and keep at room temperature until centrifugation, which must be done within 4 hours of collection. must.

3. Blood samples are centrifuged in a horizontal rotor (swing-out head) at 1500 g (±100 g) for 10 minutes at room temperature. Do not use the brake to stop centrifugation.

4. Supernatants (plasma) are carefully aspirated at room temperature and pooled in centrifuge tubes. Make sure the cell layer is not disrupted and the cells are not dislodged.

5. Carefully transfer a 4 mL aliquot of supernatant to a cryovial tube.

6. Close the cap tightly and place on ice immediately after dispensing. This step should be completed within 1 hour of centrifugation.

7. Ensure that cryovials are properly labeled with relevant information, such as details of additives contained in the blood.

8. Specimens can be stored frozen at -20°C for up to 48 hours before being transferred to a -80°C freezer.

[II. Preparation of synthetic process control DNA]
A complementary strand of methylated zebrafish DNA having the sequence shown below is synthesized using standard DNA synthesis methods such as phosphoramidite addition, incorporating 5-methyl C bases at the positions indicated. The synthetic strands are annealed to generate double-stranded DNA fragments that are used as process controls.

A. Annealing and preparation of enriched zebrafish (ZF-RASS F1 180mer) synthetic process controls. Lyophilized single-stranded oligonucleotides are reconstituted in 10 mM Tris, pH 8.0, 0.1 mM EDTA at a concentration of 1 μM.

2. Make a 10× annealing buffer of 500 mM NaCl, 200 mM Tris-HCl pH 8.0, and 20 mM MgCl ₂ .

3 _. Anneal the synthetic strands.

In a total volume of 100 μL, combine equimolar amounts of each single-stranded oligonucleotide with 1× annealing buffer, eg, as shown in the table below.

4. The annealing mixture is heated to 98° C. for 11-15 minutes.

5. Remove the reaction tube from the heat and spin down briefly to collect the concentrate at the bottom of the tube.

6. Incubate the reaction tube at room temperature for 10-25 minutes.

7. Adjust the concentration of the zebrafish RASSF1 DNA fragment by adding 0.9 mL of fish DNA diluent (Te (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) containing 20 ng/mL bulk fish DNA), Make 1.0×10 ¹⁰ copies/μl of annealed double-stranded synthetic zebrafish RASSF1 DNA in a genomic DNA carrier.

8. Process controls are diluted with 10 mM Tris, pH 8.0, 0.1 mM EDTA to the desired concentrations, eg, as described in the table below, and stored at -20°C or -80°C.

B. Preparation of process control for 100x stock (12,000 copies/μL of zebrafish RASSF1 DNA in 200 ng/μL of bulk fish DNA). 2. Thaw the reagents. 2. Vortex and spin down the thawed reagents. Add the following reagents to a 50 mL centrifuge tube

4. Aliquot into labeled 0.5 mL tubes and store at -20°C B. Preparation of 1× stock of process control (120 copies/μL zebrafish RASSF1 DNA in 2 ng/μL fish DNA). 2. Thaw the reagents. 2. Vortex and spin down the thawed reagents. Add the following reagents to a 50 mL centrifuge tube.

4. Dispense 0.3 mL into a labeled 0.5 mL tube and store at −20° C. [III. DNA extraction from plasma]
1. After thawing the plasma and preparing the reagents and labeling the tubes, clean and prepare the biosafety cabinet for extraction.

2. Add 300 μL of proteinase K (20 mg/mL) to one 50 mL centrifuge tube for each sample.

3. Add 2-4 mL of plasma sample to each 50 mL centrifuge tube (do not vortex).

4. Mix by swirling or pipetting and let sit at room temperature for 5 minutes.

5. Add 4-6 mL of Lysis Buffer 1 (LB1) solution to bring the volume to approximately 8 mL.

LB1 prescription:
• 0.1 mL of process control of 120 copies/μL zebrafish RASSF1 DNA as above;
- 0.9-2.9 mL of 10 mM Tris, pH 8.0, 0.1 mM EDTA (eg, use 2.9 mL per 2 mL plasma sample)
3 mL of guanidine thiocyanate 4.3M with 10% IGEPAL (from a stock of 5.3 g of IGEPAL CA-630 mixed with 45 mL of guanidine thiocyanate 4.8M)
6. Invert the tube 3 times.

7. Place on a benchtop shaker (room temperature) and run at 500 rpm for 30 minutes at room temperature.

8. Add 200 μL of silica-bound beads (16 μg particles/μL) and mix by rotating.

Add 9.7 mL of Lysis Buffer 2 (LB2) solution and mix by spinning.

LB2 formulation:
- 4 mL of guanidine thiocyanate 4.3M mixed with 10% IGEPAL
・3 mL of 100% isopropanol
(Lysis Buffer 2 may be added before, after, or at the same time as the silica-bound beads)
10. Invert the tube 3 times.

11. Place on a benchtop shaker and run at 500 rpm for 30 minutes at room temperature.

12. Place the tube on the capture aspirator and run the program with magnetic capture of the beads for 10 minutes, then aspirate. This allows the beads to collect for 10 minutes before removing any liquid from the tube.

13. Add 0.9 mL of wash solution 1 (3M guanidine hydrochloride or guanidine thiocyanate, 56.8% EtOH) to resuspend the bound beads and mix by spinning.

14. Place on a benchtop shaker and run at 400 rpm for 2 minutes at room temperature. (All subsequent steps can be performed on the STARlet automation platform).

15. After repeated pipetting to mix, the contained beads are transferred to a 96 deep well plate.

16. Place plate on magnetic rack for 10 minutes.

17. Aspirate and discard the supernatant.

Add 18.1 mL of wash solution 2 (80% ethanol, 10 mM Tris, pH 8.0).

19. Mix for 3 minutes.

20. Place tube on magnetic rack for 10 minutes.

21. Aspirate and discard the supernatant.

22. Add 0.5 mL Wash 2.

23. Mix for 3 minutes.

24. Place tube on magnetic rack for 5 minutes.

25. Aspirate and discard the supernatant.

26. Add 0.25 mL Wash 2.

27. Mix for 3 minutes.

28. Place tube on magnetic rack for 5 minutes.

29. Aspirate and discard the supernatant.

30. Add 0.25 mL Wash 2.

31. Mix for 3 minutes.

32. Place tube on magnetic rack for 5 minutes.

33. Aspirate and discard the supernatant.

34. Place plate on heat block at 70° C. for 15 minutes with shaking.

35. Add 125 μL of elution buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA).

36. Incubate for 25 minutes at 65°C with shaking.

37. Place plate on magnet to collect beads and cool for 8 minutes.

38. The eluate is transferred to a 96-well plate and stored at -80°C. The volume that can be recovered/transferred is approximately 100 μL.

[IV. Quantification of DNA before bisulfite treatment]
The ACTB gene is used to measure DNA in samples and may be measured prior to further processing to assess recovery of zebrafish process controls. The following protocol is used to set up the QuARTS PCR-Flap method using 10 μL of extracted DNA.

1. A 10× oligo mix is prepared containing 2 μM each of forward and reverse primers, 5 μM each of probe and FRET cassette, and 250 μM each of deoxynucleoside triphosphates (dNTPs). (See below for primers, probes and FRET sequences)

2. A master mix is prepared as follows.

3. 10 μL of each sample is pipetted into wells of a 96-well plate.

4. Add 20 μL of master mix to each well of the plate.

5. Plates are sealed and centrifuged at 3000 rpm for 1 minute.

6. Place the plate on a real-time thermal cycler ABI 7500 or Light Cycler 480 with the following reaction conditions:

[V. Bisulfite conversion and purification of DNA]
1. Thaw all extracted DNA samples from the DNA extraction step from plasma and spin down the DNA.

2. Preparation of reagents:

3. Add 5 μL of 100 ng/μL BSA DNA carrier solution to each well of the deep well plate (DWP).

4. Add 80 μL of each sample to the DWP.

5. Add 5 μL of freshly prepared 1.6N NaOH to each well of DWP(s).

6. Mix carefully to avoid air bubbles by pipetting with a pipette set to 30-40 μL.

7. Incubate at 42°C for 20 minutes.

8. Add 120 μL of BIS SLN to each well.

9. Incubate at 66° C. for 75 minutes with mixing for the first 3 minutes.

10. Add 750 μL of BND SLN.

11. Pre-mix the silica beads (BND BDS) and add 50 μL of silica beads (BND BDS) to the wells of the DWP.

12. Mix on heater shaker at 1,200 rpm, 30° C. for 30 minutes.

13. Collect the beads on the plate magnet for 5 minutes, then aspirate and discard the solution.

Add 14.1 mL of wash buffer (CNV WSH), then move the plate to a heater shaker and mix for 3 minutes at 1,200 rpm.

15. Collect the beads on the plate magnet for 5 minutes, then aspirate and discard the solution.

16. Add 0.25 mL of wash buffer (CNV WSH), then move plate to heater shaker and mix for 3 minutes at 1,200 rpm.

17. Collect the beads on a plate magnet, then aspirate and discard the solution.

18. Add 0.2 mL of desulfonation buffer (DES SLN) and mix for 7 minutes at 30° C. and 1,200 rpm.

19. Collect the beads on the magnet for 2 minutes, then aspirate and discard the solution.

20. Add 0.25 mL of wash buffer (CNV WSH), then move plate to heater shaker and mix for 3 minutes at 1,200 rpm.

21. Collect the beads on the magnet for 2 minutes, then aspirate and discard the solution.

22. Add 0.25 mL of wash buffer (CNV WSH), then move plate to heater shaker and mix for 3 minutes at 1,200 rpm.

23. Collect the beads on the magnet for 2 minutes, then aspirate and discard the solution.

24. Transfer the plate to a heater shaker to dry and incubate at 70° C. for 15 minutes with mixing at 1,200 rpm.

25. Add 80 μL of elution buffer (ELUBFR) across all samples of DWP.

26. Incubated at 65° C. for 25 minutes with mixing at 1,200 rpm.

27. Manually transfer the eluate to a 96-well plate and store at -80°C.

28. The volume that can be recovered/transferred is about 65 μL.

[VI. QuARTS-X multiplex flap method for detecting and quantifying methylated DNA]
A. Preparation for multiplex PCR (mPCR):
1. A 10× primer mix containing forward and reverse primers for each methylation marker of interest is prepared to a final concentration of 750 nM each. 10 mM Tris-HCl, pH 8, 0.1 mM EDTA is used as the diluent, as described in the Examples above.

2. Prepare 10× multiplex PCR buffer containing 100 mM MOPS, pH 7.5, 75 mM MgCl2, 0.08% Tween20, 0.08% IGEPAL CA-630, 2.5 mM dNTPs.

3. A multiplex PCR master mix is prepared as follows.

4. Thaw DNA and spin down plate.

5. Add 25 μL of master mix to 96 well plate.

6. Transfer 50 μL of each sample to each well.

7. Seal plate with aluminum foil seal (do not use strip caps)
8. Place lid in heated thermal cycler and cycle using the following profile for about 5-20 cycles, preferably about 10-13 cycles.

9. After thermal cycling is complete, a 1:10 dilution of amplicons is performed as follows.

a) Transfer 180 μL of 10 mM Tris-HCl, pH 8, 0.1 mM EDTA to each well of a deep well plate.

b) Add 20 μL of amplified sample to each of the filled wells.

c) Repeat pipetting using a fresh tip and a 200 μL pipettor to mix the diluted sample (be careful not to generate aerosols).

d) Seal the diluted plate with a plastic seal.

e) Centrifuge the diluted plate at 1000 rpm for 1 minute.

f) Seal the remaining undiluted multiplex PCR product with a new aluminum foil seal. Place at -80°C.

[B. QuARTS method with multiplex amplified DNA:]
1. Thaw the fish DNA dilution (20 ng/μL) and use it to dilute the plasmid calibrators required for the assay (see, eg, US Patent Application No. 15/033,803, incorporated herein by reference). do. Use the table below as a guide for dilution.

2. Prepare 10x triple QuARTS oligomixes using the table below for markers A, B, and C (e.g., markers of interest, and working and internal controls such as β-actin or B3GALT6 (e.g., see See US patent application Ser. No. 62/364,082, incorporated herein)).

For example, detection of bisulfite-treated β-actin, B3GALT6, and zebrafish RASSF1 markers could be used.

3. Prepare the QuARTS flap method master mix using the table below.

4. Using a 96-well ABI plate, pipette 20 μL of QuARTS master mix into each well.

5. Add 10 μL of appropriate calibrator or diluted mPCR sample.

6. Seal the plate with an ABI plastic clear seal.

7. Centrifuge the plate at 3000 rpm for 1 minute.

8. Place the plate into an ABI thermal cycler programmed to run the following thermal protocol, then start the instrument.

An aliquot (eg, 10 μL) of diluted amplified DNA is used in the QuARTS PCR-Flap method, eg, as described above. US Patent Application No. 62/249,097, filed October 30, 2015; See also filed No. 15/335,096 and PCT/US16/58875 filed Oct. 26, 2016.

[Selection and testing of methylation markers for detecting colon cancer in plasma]
Reduced Representation Bisulfite Sequencing (RRBS) data were obtained on tissues from 19 colon cancer patients, 19 polyp patients, 19 healthy subjects, and 19 DNA extracted from the buffy coat of healthy subjects. It is a thing.

After alignment to in silico bisulfite converted versions of the human genome sequence, the average methylation at each CpG island was calculated for each sample type (i.e. tissue or buffy coat) and the marker regions were identified as follows: Selected based on criteria.

• Regions were selected to be 50 base pairs or longer.

• In the design of the QuARTS flap method, regions were selected to have at least one methylated CpG in each of a) probe region, b) forward primer binding region, and c) reverse primer binding region. For forward and reverse primers, the methylated CpG is preferably close to the 3' end of the primer, but not at the 3' terminal nucleotide. An exemplary flap endonuclease method oligonucleotide is shown in FIG.

• Preferably, the buffy coat methylation at any CpG within the region of interest is less than 0.5%.

• Preferably, the methylation of cancer tissue within the region of interest is greater than 10%.

• For assays designed for tissue analysis, the methylation of normal tissue within the region of interest is preferably less than 0.5%.

GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; FD; PKIA; PPP2R5C TBX15; TSPYL5; VAV3; and ZNF671 were selected and the QuARTS flap method for them was designed as shown in FIG.

Twenty-seven markers selected from the tissue screening results were triplicated with the assay for bisulfite-converted β-actin and used to test DNA isolated from plasma samples as described above. Plasma CEA protein was measured using the Luminex Magplex assay according to the manufacturer's protocol (Luminex Corp.). DNA from 2 mL plasma samples (89 cancer and 95 normal) was extracted and eluted in 125 μL. A 10 μL aliquot of extracted DNA was used in the QuARTS method to detect β-actin and zebrafish synthetic targets. An 80 μL aliquot of DNA was converted with bisulfite as described in Example 1 and eluted at 70 μL.

Using forward and reverse primers for the targets shown in FIG. was used to detect markers.

Based on the sensitivity of the individual markers, the following 12 methylation markers were selected for further analysis: VAV3, ZNF671, CHST2, FLI1, JAM3, SFMBT2, PDGFD, DTX1, TSPYL5, ZNF568, GRIN2D, QKI.

All 12 markers were pre-amplified together using the primers shown in Figure 1 for these markers. The pre-amplified material was analyzed with the multiplexed QuARTS method as described in Example 1 using the primers and probes shown in FIG. Multiplexed assays were grouped as follows.

In addition to the above, CEA protein in the same samples was measured as described above. Data and results are shown in FIGS. The sensitivities of the individual markers at 90% specificity were as follows.

At the individual cut-off value of 95% for each individual marker, we obtained the following final sensitivities for using the pooled dataset.

The overall specificity of the assay was (88/95=92.6%).

Thus, the combination of these 12 markers and the CEA protein resulted in a sensitivity of 67% (88 of 95 cancers) and a specificity of 92.6% for all cancer tissues tested. Assayed in tissues, this panel of methylated DNA markers achieves a very high degree of discrimination for any type of colon cancer while remaining negative in normal colon tissue. Assays for this panel of markers can also be applied to tests using blood or bodily fluids, eg, for colon cancer screening.

Multiple target sequences reporting to one dye In the following experiments related to the amplified flap cleavage method, which is configured to have multiple target-specific primary cleavage reactions, reporting to a single FRET cassette results in a fluorescence signal is generated in a single dye channel. Different targets to be detected can be, for example, different markers or genes, different mutations, or different regions of a single marker or a single gene. Example 3 relates to detecting methylation of multiple different markers associated with cancer, e.g., colon cancer, using a single FRET cassette and dye channel; It relates to detecting multiple regions within a single marker using cassettes and dye channels.

Reagents used in the following experiments:

[Multiple markers reporting to one dye]
As noted above, in some embodiments it is desirable to have a higher number of markers in a single reaction using a single FRET cassette and single dye channel. In developing tests to detect multiple markers reporting to a single FRET cassette and a single dye, markers with similar reaction efficiencies (i.e., per copy of target, which gave the same amount of detectable signal) were selected. An advantage of combining detection methods with the same or similar reaction efficiencies is that any individual calibrator for one of the assays may be used as a calibration standard for any detection method with matching efficiencies.

Three markers were selected for testing in a multiple marker/single dye system (SFMBT2, VAV3, and CHST2). These target DNAs were mixed in an oligonucleotide mix, where the assay oligonucleotides for all three markers were configured to report to the same FRET cassette and therefore to the same dye (FAM). . Three disease-associated markers reporting FAM dyes and reagents detecting bisulfite-converted β-actin DNA as a control were combined in the same reaction (using the QUASAR670 FRET cassette).

Testing with plasmid calibrators was performed and the data showed that the use of multiple markers reporting to a single dye was an efficient way to overcome the need to run the markers in separate wells.

[Example 3.1]
In the QuARTS flap endonuclease method of multiple different markers to be performed in one multiplex reaction, reporting to a single FRET cassette, each individual marker was added to allow the reaction to reach equilibrium when combined in a multiplex configuration. Reaction efficiency was analyzed first. Assays were performed to determine the assay performance of three selectable markers (VAV3, SFMBT2_897 and CHST2_7890) reporting to one dye (FAM) duplexed with bisulfite-converted β-actin (BTACT). At the same time, it was configured to generate a signal reporting to the Quasar 670 channel.

The assay was also configured to determine whether each marker showed similar QuARTS method performance (slope/intercept/copy number) when the three markers reported to the same channel (FAM).

An oligonucleotide mix was prepared containing reagents to detect all three methylation markers reported in the FAM FRET cassette. The oligonucleotide mix contained reagents to detect BTACT as a control reporting to Quasar670. This oligonucleotide mix was tested against plasmid targets containing individual plasmids containing BTACT DNA and marker target DNA. Calculations were performed to determine if the calibrator curve for one marker could be used to accurately quantify the other marker. All reactions were performed in quadruplicate.

protocol:
Dilutions of stock plasmids containing one marker plasmid and one BTACT control plasmid each (see Reagent Table above) were added to a dilution of 10 mM Tris, 0.1 mM EDTA containing 20 ng/μL fish DNA as follows: Prepared as follows.

The following dilutions were prepared from the three plasmid mixtures prepared above:

A 10× oligonucleotide mix containing assay oligonucleotides (primers, probes, FRET cassette) and dNTPs was made as follows:

QuARTS flap endonuclease method reaction setup:
Prepare the master mix for the QuARTS amplification reaction as follows:

[The reaction was set up as follows:]
Using a multichannel pipette, pipette 20 μl of master mix into a 96-well QuARTS plate Add 10 μl of sample Seal the plate and centrifuge at 3000 rpm for 1 minute.

Run the plate on a LightCycler 480 using the following conditions and detect on the FAM, HEX and Quasar 670 channels: 465-510 nm, 533-580 nm, and 618-660 nm.

[result:]
Strand count using VAV3/BTACT plasmid calibrator standard curve:

Strand count using SFMBT2_897/BTACT plasmid calibrator standard curve:

Strand count using CHST2_7890//BTACT plasmid calibrator standard curve:

These data show that:
No cross-reactivity or background signal occurred when markers and controls were amplified and detected together;
- Cp values were similar for both CHST2_7890 and VAV3;
- The Cp value of SFMBT2_897 appears at an earlier cycle than CHST2_7890 and VAV3, indicating that it is faster in the QuARTS process reaction;
- Due to the fast reaction of SFMBT2_897, the combination of SFMBT2_897 calibrator and oligonucleotide mix underestimates the number of strands present for VAV3 and CHST2_7890;
- The CHST2_7890 calibrator gives calculated values of VAV3 that show assay performance equivalent to the assay response of CHST2_7890, but overestimates the amount of SFMBT2_897;
- The VAV3 calibrator gives a calculated value of CHST2_7890 that indicates assay performance equivalent to the VAV3 assay reaction, but generates an overestimation of the amount of SFMBT2_897; It is necessary to reduce the performance of the method to match the performance of both targets SFMBT2_897 and CHST2_7890.

[Experiment 3.2]
The above data showed that the SFMBT2_897 assay reaction produced a higher signal, indicating that the reaction is fast. In order to multiplex these markers, the SFMBT2_897 assay must be refined to match the efficiency of the slower assay (ie, match the signal output of both the VAV3 and CHST2_7890 assays). The following experiments tested whether this could be achieved by concentration modification of the forward primer of SFMBT2_897.

protocol:
Assays were performed as described in Experiment 3.1 above. 10× oligonucleotide mixes were constructed containing the components listed above, but with reduced amounts of the SFMBT2 — 897 forward primer for final assay concentrations of 200 nM (as in experiment 3.1), 100 nM, or 50 nM. All other assay primer concentrations were 200 nM in the final reaction mixture and the Light Cycler protocol was similar to that described in Experiment 3.1. Results indicated that decreasing the SFMBT2_897 forward primer concentration did not appear to have any effect on the slope or intercept of the signal curve, which reflects PCR efficiency (data not shown). Furthermore, the Cp value was unchanged, so the calculated chain numbers for SFMBT2_897 did not match those of the other marker targets.

[Experiment 3.3:]
The following experiments tested whether concentration modification of the SFMBT2_897 probe reduced the efficiency of the SFMBT2_897 assay, consistent with the signal output of the CHST2_7890 and VAV3 amplification reactions. Assays were performed as in Experiment 3.1 above. SFMBT2 — 897 probe oligonucleotide, and both CHST2 — 7890 and VAV3 probes, present at 500 nM (described in Experiment 3.1), 10× An oligonucleotide mix was constructed. The Light Cycler protocol was similar to that described in Experiment 3.1.

result:
Strand count using VAV3/BTACT plasmid calibrator standard curve:

Strand count using SFMBT2_897/BTACT plasmid calibrator standard curve:

Strand count using CHST2_7890/BTACT plasmid calibrator standard curve:

result:
These data show that adjusting lower probe concentration slightly increases the intercept and slightly increases the PCR efficiency (%). Cp values are also increased, so chain number calculations yield similar results to those calculated using other markers in the calibration standards.

At a SFMBT2_897 probe concentration of 250 nM, similar chain number calculations were obtained for the three markers, with the chain number for SFMBT2_897 slightly higher than the other markers. A probe concentration of 50 nM resulted in calculations that slightly underestimated the strand number, but some improvement was obtained. Therefore, a SFMBT2_897 probe concentration of 200 nM probe was chosen for subsequent studies.

[Experiment 3.4:]
This experiment tested the standard conditions described in Experiment 3.1 (all marker probes used at 500 nM) for a 10× oligonucleotide mix providing the SFMBT2 — 897 probe at 200 nM and the other probes at 500 nM. This experiment also uses multiple targets to determine if there is an additive effect of reacting in a single reaction, all of which use the same FRET cassette and dye to report a signal. Single plasmid targets, double and triple combinations of plasmid targets were used, and every target combination included the BTACT target as a control.

Plasmid dilution for one marker and control:
For reactions with single marker plasmids and BTACT control plasmids, a mixture was made containing 1.00E+04 copies/μL of each plasmid in a diluent of 10 mM Tris, 0.1 mM EDTA containing 20 ng/μL fish DNA. Marker plasmids are listed in the Reagent Table in Experiment 3.1. The targets in the plasmid mixture were as follows.

-SFMBT2_897/BTACT
-CHST2_7890/BTACT
-VAV3/BTACT
Plasmid dilution for two markers and controls:
In reactions with two marker plasmids and a BTACT control plasmid, a mixture was made containing 1.00E+04 copies/μL of each plasmid in a diluent of 10 mM Tris, 0.1 mM EDTA containing 20 ng/μL fish DNA. The targets in the plasmid mixture were as follows.

-SFMBT2_897/VAV3/BTACT
-CHST2_7890/VAV3/BTACT
-CHST2_7890/SFMBT2_897/BTACT
Plasmid dilution for 3 markers and controls:
In reactions using the three marker plasmids and the BTACT control plasmid, a mixture was made containing 1.00E+04 copies/μL of each plasmid in a diluent of 10 mM Tris, 0.1 mM EDTA containing 20 ng/μL fish DNA. The plasmid mixture was as follows.

-VAV3/CHST2_7890/SFMBT2_897/BTACT
Each of the plasmid mixtures was used to prepare solutions with 1.00E+03 copies/μL and 1.00E+02 copies/μL of each plasmid in fish DNA diluent.

The 10× oligonucleotide mix contained primers and probes for all three markers and primers and probe for the BTACT control plasmid, with probe concentrations that yielded 500 nM probe in each QuARTS method reaction, whereas for the SFMBT2_897 probe, each An amount was used to give a concentration of 200 nM SFMBT2_897 probe in the reaction. The QuARTS method components were mixed and the assay was performed on the Light Cycler as described in Experiment 3.1.

[result:]
Strand count using VAV3/BTACT plasmid calibrator standard curve:

Strand numbers for single marker and control plasmids:

Strand numbers for two markers and a control plasmid:

Strand numbers for 3 markers and control plasmid:

Strand count using SFMBT2_897/BTACT plasmid calibrator standard curve:

Strand numbers for single marker and control plasmids:

Strand numbers for two markers and a control plasmid:

Strand numbers for 3 markers and control plasmid:

Strand count using CHST2_7890/BTACT plasmid calibrator standard curve:

Strand numbers for single marker and control plasmids:

Strand numbers for two markers and a control plasmid:

Strand numbers for 3 markers and control plasmid:

These data confirm the results presented in Experiment 3.2 and show that adjusting the SFMBT2_897 probe concentration down to 200 nM aligned the efficiency of this assay reaction with that of the reaction for detection of VAV3 and CHST2_7890. showing. These data also show that when multiple targets in a reaction report signal to the same FRET cassette and dye channel, the results show an additive effect on the amount of fluorescent signal produced in that reaction. Surprisingly, no increase in background or cross-reactivity was observed. The data further indicate that the strand numbers of SFMBT2_897 and CHST2_7890 DNA calculated from the data at the lower end of the curves overestimated the amount actually added to these reactions when a dilution series of VAV3 was used as a calibration standard. evaluate. The VAV3 amplification curve is more erratic at the lower end of the standard curve, resulting in overestimation of the strand numbers of other markers.

[Experiment 3.5:]
In this experiment, the VAV3 marker probe and primer concentrations were adjusted to reduce overestimation for low level targets when the VAV3 calibrator curve was used as the reference curve for DNA concentration calculations.

For the VAV3 calibration curve, the dilution series with the VAV3 plasmid in combination with the BTACT plasmid was similar to that described in experiment 3.4. Plasmid dilutions with all three markers and a BTACT control were used.

A 10× oligonucleotide mix was made containing primers and probes for all three markers and primers and probes for the BTACT control plasmid to give the concentrations of primers and probes indicated below.

1. VAV3 (400 nM primer)/SFMBT2_897 (200 nM probe)/CHST2_7890/BTACT
2. VAV3 (750 nM probe)/SFMBT2_897 (200 nM probe)/CHST2_7890/BTACT
3. VAV3/SFMBT2_897 (200 nM probe)/CHST2_7890/BTACT
Final reaction concentrations for all other primers were 200 nM for each primer and final reaction concentrations for all other probes were 500 nM for each probe, except for the variations in primer and probe concentrations indicated above. The QuARTS method reactions were mixed and the assay was performed on the Light Cycler as described in Experiment 3.1. VAV3 calibration reactions are shown in Figures 5A-5D. FIG. 5E compares the fluorescence curves of reactions with 200 strands of target DNA measured under each condition.

Although both condition modifications improve the slope of the low calibrator of the VAV3 assay, these conditions produce the same signal as the single-marker oligonucleotide mix. The data show that the single marker mix does not have the problem of overestimating the strand number at the bottom end of the standard curve. Based on these data, 400 nM of each VAV3 primer and 500 nM of probe was selected for studies testing the assay in clinical samples.

[Experiment 3.6]
In this experiment, a sample configuration of multiple markers/one dye is tested in human plasma clinical samples. Plasma samples were pre-tested using the standard 1-marker:1-dye method as described in Example 2. The same samples were tested again using an oligonucleotide mix with VAV3, SFMBT2_897 and CHST2_7890 reporting in one fluorescence channel (FAM).

In Example 2, DNA was prepared from a series of plasma samples and target DNA was amplified by the QuARTs method. The amplicon material generated from samples 105-120 (see Figure 3) in Example 2 was diluted 1:10 and tested using the 3-target/1 control oligonucleotide mix (experiment 3.5 above). .

Dilutions of single marker/BTACT plasmid calibrators were similar to those described in experiment 3.1. configured to generate a reaction with 400 nM of each VAV3 primer and 200 nM of SFMBT2 — 897 probe, containing primers and probes for all three markers and BTACT control DNA, respectively, as described in Experiment 3.5; , with 200 nM of all other primers and 500 nM of all other probes, a 10× oligonucleotide mix was used. The QuARTS method was mixed and the assay was performed on the Light Cycler as described in Experiment 3.1. Each reaction was performed in duplicate. The results are shown in FIG.

Raw data from clinical samples 105-120 tested with these markers (from FIG. 3) are summarized in FIG. 6A. Results using a triplicate assay in which all markers report to a single FRET cassette/single dye are summarized in Figure 6B.

The target strand number for each sample was calculated separately using calibration curves for each of the three different markers. The chain values obtained were similar regardless of which standard curve was used. Moreover, the strand number of each sample using the single-dye configuration approximated the total strand number of this set of markers measured in Example 2 using separate FRET cassettes and separate dye channels. Furthermore, samples that had zero strands detected, i.e., samples that did not generate a signal in the experiment of Example 2, remained zero when using multiple markers reporting in a one-dye configuration. shows that the background signal does not increase when the multiplexed reaction reports to 1 FRET cassette/single dye channel.

These results demonstrate that detection sensitivity can be enhanced by using one FRET cassette and multiple different target sites, e.g., multiple different marker genes reporting to the same dye, and multiplex combinations It also shows that you are not limited by the number of dye channels available for signal detection. Furthermore, use of this method does not require having a single dye per reaction well. For example, an assay can be configured with three (or more) markers reporting on a first dye (e.g., FAM) and three (or more) markers reporting on a second dye (e.g., HEX). could be configured to double the number of markers that can be tested in a single reaction for a single preparation of nucleic acid sample. Additional dye channels may be used for additional sets of markers and/or one or more internal control targets.

[Multiple regions of markers reporting to one dye]
For the three methylation markers VAV3 (877), SFMBT2 (897), and CHST2 (7890) that showed low or no chain number in normal plasma using the methods described herein above, Additional QuARTS method oligonucleotide sets targeting other regions of the interior were designed and tested to detect additional regions of the marker in the same reaction and report to the same dye channel, thereby increasing the signal for each marker. It was investigated whether increasing the noise-to-noise ratio would increase the sensitivity of the assay, eg, in detecting cancer.

For each of these markers, we identified two distinct regions that were determined by RRBS to have differential methylation between cancer and normal tissue. Those areas are:

- VAV3 region 877: chr1: 108507618-108507675
- VAV3 region 11878: chr1: 108507406 to 108507499
- SFMBT2 region 895: chr10: 7452337-7452406
- SFMBT2 region 897: chr10: 7452865-7452922
-CHST2 region 7890: chr3: 142838847 to 142839000
- CHST2 region 7889: chr3: 142838300 to 142838388
[Experiment 4.1]
The CHST2 regions (7889 and 7890) reporting to the HEX dye were tested in both individual and pooled responses to assess if there was any synergy between the two regions when pooled. The calibrator plasmid containing the CHST2 insert was diluted as described in Experiment 3.1 to give a serial dilution of 1E4-1E0 copies per μL. For individual detection of region 7889, the assay reaction contained forward and reverse primers and arm 1 probe for CHST2_7889, arm 1 HEX FRET cassette, and arm 3 probe for primers and BTACT controls, along with arm 3 Quasar670 FRET. A cassette was included. For individual detection of region 7890, the assay reaction contained forward and reverse primers and arm 1 probe for CHST2_7890, arm 1 HEX FRET cassette, and arm 3 probe for primers and BTACT controls, along with arm 3 Quasar670 FRET. A cassette was included. Combined reactions contained a complete set of arm 1 probes and primers for both CHST2_7889 and 7890, along with BTACT detection oligonucleotides and two identical FRET cassettes.

The 10× oligonucleotide mix contained concentrations of primers and probes that resulted in 500 nM of each probe and 200 nM of each primer in each QuARTS method reaction. The QuARTS method components were mixed and the assay was performed on the Light Cycler as described in Experiment 3.1.

In the combined reaction, we found that using a single FRET cassette and having these two regions report the same dye did not result in an increase in signal. Since amplification of CHST2_7889 appeared to be substantially more efficient and dominated the signal obtained, the different reactions were modified to be more similar in efficiency, as discussed in Example 3. It is suggested that

[Experiment 4.2]
What probe concentrations are used for each pair of regions {CHST2 (7889 and 7890), SFMBT2 (895 and 897) and VAV3 (877 and 11878)} of each marker to equilibrate the kinetics between the different regions Experiments were conducted to determine if A 10× oligonucleotide mix was made and the following mixture of assay oligonucleotides was obtained at the final concentrations indicated.

The QuARTS method components were mixed and the assay was performed on the Light Cycler as described in Experiment 3.1. The average Cp values achieved under different reaction conditions are:

These data demonstrate that the Cp values of individual assays can be adjusted by varying the probe concentrations for each of the two regions of each marker, to the point where each of the five points on the calibration curve has a Cp value of less than 1. show. For the markers tested, using the following probe concentrations in the QuARTS method reactions resulted in balanced reaction efficiencies for the target region set.

[Experiment 4.3]
A new triplex reaction (see Example 2 for the original triplex reaction configuration) was designed to use the multiple regions/one dye assay configuration in a multiplexed reaction. "Pool 17" below is a listing of a set of 6 markers, co-amplified with a β-actin control and then divided into groups as indicated below and analyzed by the triple QuARTS method. Pool 17+MR-OD is configured to contain assay configurations of multiple regions/one dye for each of the SFMBT2, VAV3, and CHST2 markers. The assay designs for JAM3, ZNF671 and ZNF568 were as shown in FIGS. The three-letter or four-letter abbreviation for each grouping of pools is the initial letter of each gene name with an A indicating the β-actin control.

A new triplex formulation was prepared with a plasmid calibration dilution series containing pool 17 multiplexes containing all target regions of the above groups in a dilution series providing 2e5-2e1 of each target strand per assay reaction. tested. The final probe concentrations for SFMBT2, VAV3, and CHST2 MR-OD were similar to those described in the results of Experiment 4.2. JAM3, ZNF671, and ZNF568 marker probes and BTACT control probes were at 1 μM. All FRET cassettes were 500 nM in the final reaction mix. The QuARTS method components were mixed and the assay was performed on the Light Cycler as described in Experiment 3.1.

The performance of the triplex containing VAV3-877 and VAV-11878 is as expected, yielding approximately 2-3 fold increase in strand number over the number of targets added to the reaction, and only one region. The target was oriented as a target. However, triplex containing CHST2-7889_CHST-7890 and SFMBT2-895_SFMBT2-897 did not show the expected additive signal. Further experiments were performed using different concentrations of the CHST2-7889_CHST2-7890 and SFMBT2-895_SFMBT2-897 probes to test the multiplex QuARTS method grouped as described above. Within triplex format, CHST2_7889 and CHST2_7890 probe concentrations were modified to achieve predicted MR_OD results (i.e., results with additive values predicted for individual reactions) based on the plasmid calibration curve. was possible. However, although the SFMBT2_895 and SFMBT2_897 assays were improved using modified probe concentrations, the assays still fell below the expected level of 200% expected for detection of the two regions when used in the triplex format. A signal was generated. Nevertheless, the following corrected concentrations of probe were chosen for testing the triplex assay on plasma samples.

[Experiment 4.4]
This experiment demonstrates the effect of integrating triple QuARTS detection and multiplex pre-amplification using a multi-domain-1 dye assay design to test human plasma samples from healthy and cancer patients. examined. Experiments compared the detection of the 13 methylation markers of pool 17 (plus ZF_RASSF1, a process control) to detection using the pool 17+MR_OD configuration in 63 normal and 12 colon cancer plasma samples. The markers of Pool 17 were co-amplified together in a pre-amplification followed by detection of pre-amplified DNA as detailed in Example 1 in the grouped reactions listed below.

The triplex name includes the initial letter of each marker included, plus an "A" for the β-actin control. In the right column, repeated letters (eg, "JSSA") in triplex names indicate that one marker was tested in two different regions.

DNA was isolated from plasma samples as described in Example 1. Bisulfite conversion, multi-strand pre-amplification, and QuARTS procedures were performed as described in Example 1 on multiplex amplified DNA. Prior to bisulfite conversion, an aliquot of isolated DNA was set aside to test the KRAS 38A and 35C mutations on unconverted DNA. The amplification primers and detection probes used for each marker are shown in FIGS.

As shown below, the logistic linear regression fit using the number of strands per reaction for VAV3, SFMBT2, CHST2, and ZNF671 was better than that of the QuARTs method when QuARTs were used in combination with MR_OD (multiple region_1 dye). It showed considerable superiority compared to the standard configuration. In these analyses, the marker ZNF671 was the major contributor to the detection results and was included in the logistic fitness of both QuARTs alone and QuARTs+MR_OD. Mutations of KRAS 38A and 35C in unconverted DNA were also tested as described above.

The following sensitivities and specificities were obtained using multiplex preamplification in standard triplex assays.

Sensitivity and specificity using the multi-region/one-dye configuration were as follows.

Even at small sample sizes, this multi-region-to-single-dye configuration (FRET cassette) represents a substantial improvement in sensitivity, although some loss of specificity may occur.

Although DNA isolated from plasma samples was detected in this example, the use of the markers in this panel, and the multi-stranded QuARTS method modified as described above, is applicable to studies using faeces or other blood or body fluids. and is used, for example, in screening for colon and other cancers.

All publications and similar materials cited in this application, including, but not limited to, patents, patent applications, articles, books, papers, and Internet web pages, are incorporated by reference in their entirety for any purpose. explicitly incorporated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong. If the definitions of terms in the incorporated references differ from the definitions set forth in the present teachings, the definitions set forth in the present teachings shall control.

Various modifications and variations of the described compositions, methods, and technical uses will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. be. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in pharmacology, biochemistry, medicine, or related fields are intended to be within the scope of the following claims. .

Claims (19)

A method of screening for colon tumors in a plasma sample obtained from a human subject, comprising:
a) extracting DNA from said plasma sample, wherein extracting DNA from said plasma sample comprises adding to said plasma sample a composition comprising methylation process control DNA; The DNA comprises a zebrafish rassf1 gene or a zebrafish DNA nucleotide sequence comprising a polynucleotide comprising SEQ ID NO: 177 as the sense strand and SEQ ID NO: 178 as the antisense strand,
b) assaying the extracted DNA for the amount of the methylation process control DNA to assess the efficacy of the extracted DNA from the plasma sample;
c) assaying said extracted DNA for the amount of a plurality of different methylation marker DNAs, said plurality of different methylation marker DNAs having at least one methylation selected from the group consisting of: GRIN2D; and ZNF671. FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5;
d) assaying the sample for the amount of standard nucleic acid in the extracted DNA;
e) comparing the amount of the methylation marker DNA and the amount of the standard nucleic acid in the extracted DNA to determine the methylation status of the methylation marker DNA in the sample; and f) at least one If the methylation status of the plurality of methylation marker DNAs is higher than the methylation status of the methylation marker DNA assayed in a subject without colon tumor, the subject is defined as having a colon tumor. to identify as
The above method, comprising 2. The assay of claim 1, wherein the assay comprises treating DNA obtained from the sample with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues. described method. 3. The method of claim 2, wherein the reagent is a bisulfite reagent. The method of any one of claims 1-3, wherein the standard nucleic acid comprises B3GALT6 DNA. LRRC4; OPLAH; SEP9; SLC12A8; ZNF304; DOCK2; 5. The method according to any one of 1 to 4. The method of any one of claims 1-5, wherein the method further comprises assaying the sample for carcinoembryonic antigen (CEA) protein. 7. The method of any one of claims 1-6, wherein assaying the amount of methylation marker DNA in the sample comprises determining the methylation status of one base of the methylation marker DNA. 8. The method of any one of claims 1-7, wherein assaying the amount of methylation marker DNA in the sample comprises determining the degree of methylation at multiple bases of the methylation marker DNA. The amount of methylation marker DNA in said sample indicates the methylation status of the methylation marker gene, wherein said methylation status of said methylation marker gene is:
- an amount of said methylation marker gene that is increased over the amount of said methylation marker gene in a normal sample - a different pattern of methylation of said marker gene compared to the methylation pattern of said methylation marker gene in a normal sample A method according to any one of claims 1 to 8, comprising at least one. Said assay comprises using the polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass separation, or target capture, flap endonucleases, multiplex amplification, and said at least one Determination of the amount of the two methylation marker DNA is performed by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease, PCR-flap, and bisulfite genome sequencing PCR. a) at least one plurality of oligonucleotides, at least a portion of which specifically hybridizes to GRIN2D methylation marker DNA, or at least a portion of which specifically hybridizes to ZNF671 methylation marker DNA FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; at least one further oligonucleotide of said plurality of oligonucleotides that specifically hybridizes to
b) at least one standard oligonucleotide, at least a portion of which specifically hybridizes to the standard nucleic acid;
c) a methylation process control comprising a zebrafish DNA nucleotide sequence at least a portion of which comprises the zebrafish rassf1 gene or a polynucleotide comprising SEQ ID NO: 177 as the sense strand and SEQ ID NO: 178 as the antisense strand at least one process control oligonucleotide that specifically hybridizes to DNA;
and optionally d) one or more of the following - a bisulfite - a reagent for detecting CEA protein - a solid support,
- one or more capture reagents,
A kit for use in screening for colon tumors based on methylation status in plasma samples obtained from human subjects , comprising: 12. The kit of claim 11, wherein said standard nucleic acid comprises B3GALT6 DNA. 13. The kit of claim 11 or 12, wherein said solid support is magnetic beads. The kit of any one of claims 11-13, wherein said one or more capture reagents comprise oligonucleotides complementary to said plurality of methylation marker DNAs. The kit of any one of claims 11-14, wherein said one or more capture reagents comprise oligonucleotides complementary to said standard nucleic acid. The kit of any one of claims 11-15, wherein said at least one oligonucleotide in said plurality of oligonucleotides is selected from one or more of a nucleic acid primer pair, a nucleic acid probe, and an invasion oligonucleotide. . target DNA extracted from a plasma sample of a human subject suspected of having a colon tumor and treated with a reagent that selectively modifies unmethylated cytosine residues in the DNA to produce modified residues; , a reaction mixture comprising a complex with an oligonucleotide, wherein said oligonucleotide specifically hybridizes to said target DNA, said oligonucleotide comprising one or more fluorophore and flap sequences; A composition, optionally comprising, wherein said complex is selected from the group consisting of:
i) a complex comprising GRIN2D target DNA that specifically hybridizes to a GRIN2D-specific oligonucleotide; and ii) a complex comprising ZNF671 target DNA that specifically hybridizes to a ZNF671-specific oligonucleotide.
The composition further comprises:
iii) a complex comprising a further target DNA selected from the group consisting of VAV3; CHST2; FLI1; JAM3; specifically hybridizes to nucleotides,
The composition further comprises a zebrafish rassf1 gene or a zebrafish DNA nucleotide sequence comprising a polynucleotide comprising SEQ ID NO: 177 as the sense strand and SEQ ID NO: 178 as the antisense strand. ,Composition. 18. The composition of claim 17, further comprising one or more of a FRET cassette, a FEN-1 endonuclease; and a thermostable DNA polymerase. 19. The composition of claim 17 or 18, further comprising a standard nucleic acid comprising B3GALT6 DNA, and an oligonucleotide that specifically hybridizes to said B3GALT6 DNA.

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